• electrical properties of DNA polymerase?
• enzymatic synthesis of nucleic acids
• chemoenzymatic synthesis
• chemical oligonucleotide
• in vivo DNA synthesis
• in vivo gene synthesis

Enzymatic DNA synthesis

http://groups.google.com/group/enzymaticsynthesis

http://github.com/kanzure/enzymaticsynthesis

Mechanisms of attack:

(1) template DNA/RNA
(2) selected dNTP
    - electronic control to force a specific dNTP to be selected

Direct control of polymerase

1) template shams
2) forced misincorporation

crazy ideas:

1. electronic control of polymerase
2. nucleotide gun made out of a nanotube pointed at the finger domain of dna polymerase
3. single-polymerase water droplet & add in a single dNTP at a time
4. physical display of dNTP as template for current base addition (i.e., on a stick)
5. a protein that can undergo conformational changes that polymerase thinks represents the template strand
6. pull/push a template through DNA polymerase to control which dNTP it should be selecting for
7. protein-template DNA polymerase, where the polymerase itself has a giant protein that enzymatically encodes dNTP information
8. graph grammar rules of valid thumb/fingers/pad/palm domains of polymerases to generate a library of possible polymerase variants
9. mechanical pressure on polymerase
10. ultrasound

mRNA polyadenylation (addition of adenosine monophosphates to an mRNA fragment)

"Primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand without an initial RNA or DNA primer (for temporary DNA elongation)."

What is the actual change in DNA polymerase that decides whether a base should be accepted or not? Is primase template-dependent?

replicase - RNA-dependent RNA polymerase

In the transfer RNA (tRNA) CCA-adding enzyme, both the tRNA backbone and the protein contribute to the specificity of the incoming nucleotide (24). Eukaryotic translesion synthesis polymerases thus use a variety of means of DNA polymerization, which include Watson-Crick base-pairing by Pols n (25) and kappa (26), Hoogsteen base-pairing by Pol?? (18, 22), and protein template-directed synthesis by Rev1.

primer-template slippage

tdt

EC2.7.7.31 -- terminal deoxynucleotidyltransferase TDT

http://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase

http://www.rcsb.org/pdb/explore/explore.do?pdbId=1jms

http://www.rcsb.org/pdb/explore/explore.do?pdbId=1coe

author: Basu (again)

(02:43:38 PM) Cathal Garvey: Here, this enzyme: Terminal Transferase and easily removed blocking groups on NTP washes = Easy homebrew DNA synthesis reaction? http://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase
(02:44:13 PM) Cathal Garvey: In other words, not so dissimilar to the existing method, but without requiring the chemistry of actually joining nucleotides with such care. In fact, you could probably do something like this:
(02:44:21 PM) Cathal Garvey: Oligo annealed to solid substrate
(02:44:29 PM) Cathal Garvey: Ends not phosphorylated at beginning of each step.
(02:45:09 PM) Cathal Garvey: Add a kinase to prime DNA, add NTP + TDT
(02:45:32 PM) Cathal Garvey: Interesting stuff!
(02:45:44 PM) Cathal Garvey: I love all the little methods devised by nature for this function
(02:45:49 PM) Cathal Garvey: By the way, my error there:
(02:46:06 PM) Cathal Garvey: the method I suggest is based on the assumption that the enzyme might bind the oligo end and await an NTP,
(02:46:16 PM) Cathal Garvey: and that you can use NDPs rather than NTPs
(02:46:35 PM) Cathal Garvey: so that it won't polymerise further than a single nucleotide without a step involving kinase
(02:47:06 PM) Cathal Garvey: So you can use two enzymes and NDPs in washes, if that prior assumption regarding the enzyme holds.
(02:47:15 PM) Cathal Garvey: Which is, of course, a huge achilles heel.

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg2+ or Mn2+ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.

.. so primarily dGMP and dAMP?

• The Use of Non-natural Nucleotides to Probe Template-Independent DNA Synthesis
• Mechanisms by which human DNA primase chooses to polymerize a nucleoside triphosphate
• Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase

Analysis of diversity of nucleotide and amino acid distributions in the VD and DJ joining regions in Ig heavy chains

preference for polar residues - more likely to encourage interaction with ligand than non-polar residues (often found in loop regions in general). 
preference for the insertion of smaller residues versus larger residues (this aides in loop flexibility)

Nucleotide fill-in between the germ line V, D and J genes in the H3 loop of immunoglobulins contributes to the diversity of the antibody repertoire. This fill-in process is mediated by terminal deoxynucleotidyl transferase (TdT), which has been widely believed to insert nucleotides in a random fashion. Using a database of 2443 immunoglobulin sequences, we identified the regions of nucleotide fill-in between the V–D and D–J gene regions. We translated the fill-in nucleotides and measured the diversity within the two regions both at the nucleotide and amino acid level. We found that the nucleotide and amino acid distributions that resulted from nucleotide fill-in were in fact not random. Examination of the synonymous substitution rates of nucleotides revealed evidence suggesting that TdT plays a less significant role in generating antibody diversity than previously thought. We observed preferences for polar residues, which are more likely to encourage interaction with ligand than non-polar residues and are often found in loop regions in general. We also observed a preference for the insertion of smaller residues versus larger residues of similar biochemical properties, aiding in loop flexibility. We interpret these findings to reflect the significant influence of biochemical (i.e. folding) constraints and/or binding affinity constraints (at the cellular/selectional level) on the sequence diversity in the H3 region. These constraints act as a filter on the randomness generated by nucleotide addition by TdT, as well as other diversity generating processes such as recombination of VDJ gene segments and somatic mutation. The results of this study suggest that the antibody repertoire might be reduced from what is traditionally believed.

[81] K. Ramadan, I.V. Shevelev, G. Maga, U. Hubscher, De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase. J. Mol. Biol. 339 (2004) 395-404.

requires input of at least 3 bp

synthesizes 2 to 15 bp

TdT, like all DNA polymerases, also requires divalent metal ions for
catalysis [73,89]. However, TdT is unique in its ability to use a variety
of divalent cations such as Co2+, Mn2+, Zn2+and Mg2+. In general, the
extension rate of the primer p(dA)n (where n is the chain length from
4 through 50) with dATP in the presence of divalent metal ions is
ranked in the following order: Mg2+ N Zn2+ N Co2+ N Mn2+[73]. In
addition, each metal ion has different effects on the kinetics of
nucleotide incorporation. For example, Mg2+ facilitates the prefer-
ential utilization of dGTP and dATP whereas Co2+ increases the
catalytic polymerization efficiency of the pyrimidines, dCTP and dTTP
[90]. Zn2+ behaves as a unique positive effector for TdT since reaction
rates with Mg2+ are stimulated by the addition of micromolar
quantities of Zn2+ [90]. This enhancement may reflect the ability of
Zn2+ to induce conformational changes in TdT that yields higher
catalytic efficiencies [90]. Polymerization rates are lower in the
presence of Mn2+ compared to Mg2+, suggesting that Mn2+ does
not support the reaction as efficiently as Mg2+ [73].

[106] S.J. Benkovic, C.E. Cameron, Kinetic analysis of nucleotide incorporation and misincorporation by Klenow fragment of Escherichia coli DNA polymerase I, Methods Enzymol. 262 (1995) 257–269.

Distinct Domain Functions Regulating de Novo DNA Synthesis of Thermostable DNA Primase from Hyperthermophile Pyrococcus horikoshii

DNA structure and aspartate 276 influence nucleotide binding to human DNA polymerase β

Structures of DNA polymerase (pol) β bound to single-nucleotide gapped DNA had revealed that the lyase and pol domains form a “doughnut-shaped” structure altering the dNTP binding pocket in a fashion that is not observed when bound to non-gapped DNA. We have investigated dNTP binding to pol β-DNA complexes employing steady-state and pre-steady-state kinetics. Although pol β has a kinetic scheme similar to other DNA polymerases, polymerization by pol β is limited by at least two partially rate-limiting steps: a conformational change after dNTP ground-state binding and product release. The equilibrium binding constant,K d (dNTP), decreased and the insertion efficiency increased with a one-nucleotide gapped DNA substrate, as compared with non-gapped DNA. Valine substitution for Asp276, which interacts with the base of the incoming nucleotide, increased the binding affinity for the incoming nucleotide indicating that the negative charge contributed by Asp276 weakens binding and that an interaction between residue 276 with the incoming nucleotide occurs during ground-state binding. Since the interaction between Asp276 and the nascent base pair is observed only in the “closed” conformation of pol β, the increased free energy in ground-state binding for the mutant suggests that the subsequent rate-limiting conformational change is not the “open” to “closed” structural transition, but instead is triggered in the closed pol conformation.

Structure and mechanism of DNA polymerase β

A reexamination of the nucleotide incorporation fidelity of DNA polymerases

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a “simplified” model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.

A new paradigm for DNA polymerase specificity (Austin, Texas)

We show that T7 DNA polymerase exists in three distinct structural states, as reported by a conformationally sensitive fluorophore attached to the recognition (fingers) domain. The conformational change induced by a correct nucleotide commits the substrate to the forward reaction, and the slow reversal of the conformational change eliminates the rate of the chemistry step from any contribution toward enzyme specificity. Discrimination against mismatches is enhanced by the rapid release of mismatched nucleotides from the ternary E·DNA·deoxynucleoside triphosphate complex and by the use of substrate-binding energy to actively misalign catalytic residues to reduce the rate of misincorporation. Our refined model for enzyme selectivity extends traditional thermodynamic formalism by including substrate-induced structural alignment or misalignment of catalytic residues as a third dimension on the free-energy profile and by including the rate of substrate dissociation as a key kinetic parameter.

Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity

Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

• A unified mechanism applicable to multiple DNA polymerases
• Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue
• Mismatched and Matched dNTP Incorporation by DNA Polymerase β Proceed via Analogous Kinetic Pathways
• Kinetic analysis of correct nucleotide insertion by a Y-family DNA polymerase reveals conformational changes both prior to and following phosphodiester bond formation
• Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli
• Techniques used to study the DNA polymerase reaction pathway

• Contribution of the Reverse Rate of the Conformational Step to Polymerase β Fidelity

  ".. the ternary complex is destabilize by the presence of incorrect dNTP" By analysis of the relative magnitudes of chemistry and reverse "opening" in the presence of both matched and mismatched matched ternary complexes, this work further validates that, for Pol β, fidelity is dictated by the differences in free energy required to reach the highest energy transition state of the chemical step.

N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.

Template-Free Primer-Independent DNA Synthesis by Bacterial DNA Polymerases I Using the DnaB Protein from Escherichia coli

This is the first study to show that bacterial type I DNA polymerases (including truncted ones) can synthesize DNA in vitro in the absence of primer and template, using the product of the DnaB gene of Escherichia coli. This product is produced only when reaction mixture contains both DNA polymerase and the product of the DnaB gene. The control experiments showed that DNA synthesis is not due to the presence of oligonucleotide contaminants in reaction mixture. The reaction products may be several hundred kb in length; they are completely hydrolyzed by DNase I and partly by the nuclease S1 and contain a repetitive AT-rich sequence, in which approximately 15% of the nucleotides in which are cytosine and guanine.

De Novo DNA Synthesis by Human DNA Polymerase λ, DNA Polymerase μ and Terminal Deoxyribonucleotidyl Transferase

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3′OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3′OH end of a DNA strand, without the need of a template. Pol λ and pol μ are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol β and TdT. Here we show that pol λ, pol μ and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol λ, pol μ and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol λ is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.

The Use of Non‐natural Nucleotides to Probe Template‐Independent DNA Synthesis Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase

A light-activated DNA polymerase

The widely applied Thermus aquaticus DNA polymerase was built by the photoactivatable amino acid ortho-Nitrobenzyltyrosin instead of a tyrosine in the active center light-activated (see picture). Since the modified enzyme active after irradiation with UV light, the polymerase activity can be regulated temporally.

Die häufig genutzte Thermus-aquaticus-DNA-Polymerase wurde durch Einbau der photoaktivierbaren Aminosäure ortho-Nitrobenzyltyrosin anstelle eines Tyrosinrests im aktiven Zentrum lichtaktivierbar (siehe Bild). Da das modifizierte Enzym erst nach Bestrahlen mit UV-Licht aktiv ist, kann die Polymerase-Aktivität zeitlich reguliert werden.

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.

Light activation of transcription: photocaging of nucleotides for control over RNA polymerization

We describe the use of ATP caged with [7-(diethylamino)coumarin-4-yl]methyl (DEACM) for light-controlled in vitro transcription reactions. Polymerization is blocked when DEACM is bonded to the gamma phosphate group of the ATP molecule. Controlled light irradiation releases ATP and transcription is initiated. In order to provide full control over the process, conditions involved in substrate release, nucleotide availability after release and the effect of the released coumarin in RNA polymerization were assessed in further detail. Together, our data provide the first direct evidence of control over enzymatic polymerization of nucleic acids through light. This approach may provide researchers with a unique tool for the study of biological processes at a molecular level.

Site-specific incorporation of photofunctional nonnatural amino acids into a polypeptide through in vitro protein biosynthesis

• azobenzene-tethered dNTPs ?
• photocleavable dNTPs into polymerase
• polymerase with azobenzene/switchable amino acids at important sites

A Hydrophilic Azobenzene-Bearing Amino Acid for Photochemical Control of a Restriction Enzyme BamHI

A novel hydrophilic and negatively charged azobenzene-bearing amino acid, 4‘-carboxyphenylazophenylalanine (azoAla 1), has been designed and synthesized for investigation of the photochemical regulation of the enzyme activity. The properties of photoisomerization and thermal stability of the cis-isomer were similar to those of a commonly used phenylazophenylalanine (azoAla 2). For photochemical control of the enzyme, these two azobenzene-bearing amino acids were incorporated into the specific position at the dimer interface of a restriction enzyme BamHI. These trans-azobenzene derivatives in the BamHI suppressed the enzymatic activity, and the following photoirradiation at 366 nm induced the recovery of its activity. Although the activities of both azoAla-BamHI mutants were same level after a long time irradiation, the recovery of the activity of azoAla 1-BamHI was faster than that of azoAla 2-BamHI with a short time irradiation. This result suggests that the negatively charged carboxylate group introduced into an azobenzene moiety affects the behavior of azoAla in the protein scaffold during the trans−cis photoisomerization.

refs

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore

Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3′-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Förster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

Electronic control of DNA polymerase binding and unbinding to single DNA molecules

DNA polymerases catalyze template-dependent genome replication. The assembly of a high affinity ternary complex between these enzymes, the double strand−single strand junction of their DNA substrate, and the deoxynucleoside triphosphate (dNTP) complementary to the first template base in the polymerase active site is essential to this process. We present a single molecule method for iterative measurements of DNA−polymerase complex assembly with high temporal resolution, using active voltage control of individual DNA substrate molecules tethered noncovalently in an α-hemolysin nanopore. DNA binding states of the Klenow fragment of Escherichia coli DNA polymerase I (KF) were diagnosed based upon their ionic current signature, and reacted to with submillisecond precision to execute voltage changes that controlled exposure of the DNA substrate to KF and dNTP. Precise control of exposure times allowed measurements of DNA−KF complex assembly on a time scale that superimposed with the rate of KF binding. Hundreds of measurements were made with a single tethered DNA molecule within seconds, and dozens of molecules can be tethered within a single experiment. This approach allows statistically robust analysis of the assembly of complexes between DNA and RNA processing enzymes and their substrates at the single molecule level.

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg2+ or Mn2+ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.

--> length of oligos synthesized by tdt? 10-15bp??

Relationship Between Conformational Changes in Pol λ's Active Site Upon Binding Incorrect Nucleotides and Mismatch Incorporation Rates

The Conserved Active Site Motif A of Escherichia coli DNA Polymerase I Is Highly Mutable

Akeo Shinkai, Premal H. Patel, and Lawrence A. Loeb

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val700–Arg712 by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp705 can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild type motif A sequence. As an example, we established that the naturally occurring Ile709 has a previously undocumented role in supporting sugar discrimination.

UmuC/DinB family (e.g. DNA polymerase ). Crystal structures of representative enzymes from the first four families have been determined, revealing a common overall architecture that has been likened to a human right hand, with fingers, thumb, and palm subdomains (5–9). Although the structures of the fingers and thumb subdomains vary considerably, the catalytic palm subdomains are all superimposable (10, 11). The palm subdomain includes two conserved sequences, motif A and motif C, each harboring a catalytically essential aspartic acid residue. Essential roles of motif A in catalysis include interaction with the incoming dNTP and coordination with two divalent metal ions that are required for the polymerization reaction (12–15). Motif A begins at an anti-parallel -strand containing predominantly hydrophobic residues and is followed by a turn and an -helix. Although there is considerable variation in the amino acid sequence of the anti-parallel -strand, the sequence of the turn and helix, DYSQIELR, is nearly invariant among known prokaryotic family A polymerases (16).2

each year (30), at mutation rates of 10 5/nucleotide/division in mutators (31–33) to 10 9/nucleotide/division in nonmutators (34). Despite this conservation in nature, we show here that motif A in E. coli pol I can tolerate a substantial mutational burden, and most of the 13 constituent amino acids are replaceable, yielding highly competent variant polymerases. E. coli strains harboring active mutant pol I that were selected in a genetic complementation system are fit to replicate repetitively, both in liquid broth and on solid agar at 37 °C. These observations are consistent with biochemical data showing that the mutant proteins possess wild type-like DNA polymerase activity in vitro. We found that only one residue, the catalytically essential Asp705, was immutable; the corresponding residue in Taq pol I coordinates with the two metal ligands required for catalysis (13, 15). Substitution of Glu710 was restricted to Asp; in crystals of a closed ternary complex of T7 DNA polymerase complexed with its substrates (13), the glutamate residue equivalent to Glu710 is hydrogen-bonded with a tyrosine residue in the O-helix within the fingers subdomain. We conclude that a Asp residue at position 705 and a negative charge at position 710 are indispensable for maintaining polymerase activity; this conclusion is in agreement with the deleterious effects of the D705A and E710A substitutions on catalysis (35). Both the N- and C-terminal parts of motif A tolerated a wide spectrum of substitutions. DNA polymerase activity associated with single amino acid substitutions within the N-terminal 5 amino acid residues was as high or higher than that of the wild-type enzyme. '''These residues form part of an anti-parallel -sheet structure that is believed to accommodate the triphosphate moiety of the incoming dNTP and may be a potential target for engineering of pol I derivatives with altered properties.''' In contrast, amino acid substitutions within the C-terminal five residues tended to be associated with reduced activity.

both DNA and RNA substrates. We conclude that isoleucine at position 709 contributes to sugar discrimination by wild-type pol I and that this function may promote conservation of the wild-type motif A sequence. Based on analysis of a structural

The main difference in mutability between E. coli and Taq pol I involves a tyrosine residue. In the case of Taq pol I, Tyr611 was replaced only by the planar-ringed amino acids, Phe, His, and Trp, whereas substitutions at the corresponding Tyr706 of E. coli pol I were not similarly restricted. In the closed ternary complex of Taq pol I (15), the side chain of Tyr611 projects into a large hydrophobic pocket. We surmise that the side chains of Phe, His, or Trp may perform a space-filling function in a manner comparable with that of Tyr611, thus permitting replacement; such a function might be less important in E. coli pol I, which tolerates other replacements. Another difference in mutability is that the number of positively charged residues, especially at Ser612, Ile614, and Arg617, is greater among the Taq than the E. coli mutants. These residues may facilitate proper folding of Taq pol I while also maintaining polymerase activity at elevated temperatures (17). The optimum growth temperature for T. aquaticus is far higher than for E. coli; hence, the structure of proteins from T. aquaticus would presumably be restricted to ensure thermostability. Such structural constraints might be reflected in the restricted mutability of Tyr611 or the greater prevalence of positively charged replacements.

In conclusion, we reiterate that all DNA polymerases thus far examined appear to share a common overall architecture with superimposable catalytic palm subdomains and a common polymerase mechanism. In view of this conservation of structure and mechanism, we speculate that high mutability of motif A has also been retained throughout evolution so as to promote tolerance of a mutational burden at the polymerase active site with minimal loss of replicative capacity under conditions of changing environmental stresses.

Acknowledgments—We thank Dr. Elinor Adman for helpful discussions and Dr. Ann Blank for critical reading of the manuscript.

5. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP

Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., and Steitz, T. A. (1985) Nature 313, 762–766

blah, an image-only PDF :(

6. Meh

Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783–1790

http://www.sciencemag.org.ezproxy.lib.utexas.edu/cgi/content/abstract/256/5065/1783

A 3.5 angstrom resolution ?electron density map of the HIV-1 reverse transcriptase ?heterodimer complexed with ?nevirapine, a drug with potential for treatment of AIDS, reveals an asymmetric dimer. The polymerase (pol) domain of the 66-kilodalton subunit has a large cleft analogous to that of the ?Klenow fragment of ?Escherichia coli DNA polymerase I. However, the 51-kilodalton subunit of identical sequence has no such cleft because the four subdomains of the pol domain occupy completely different relative positions. Two of the four pol subdomains appear to be structurally related to subdomains of the Klenow fragment, including one containing the catalytic site. The subdomain that appears likely to bind the template strand at the pol active site has a different structure in the two polymerases. '''Duplex A-form RNA-DNA hybrid can be model-built into the cleft that runs between the ribonuclease H and pol active sites.''' Nevirapine is almost completely buried in a pocket near but not overlapping with the pol active site. Residues whose mutation results in drug resistance have been approximately located.

7. Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism

http://www.sciencemag.org.ezproxy.lib.utexas.edu/cgi/content/abstract/264/5167/1930

Sawaya, M. R., Pelletier, H., Kumar, A., Wilson, S. H., and Kraut, T. (1994) Science 264, 1930 –1935

Structures of the 31-kilodalton ?catalytic domain of rat DNA polymerase beta (pol beta) and the whole 39-kilodalton enzyme were determined at 2.3 and 3.6 angstrom resolution, respectively. The 31-kilodalton '''domain is composed of fingers, palm, and thumb subdomains arranged to form a DNA binding channel''' reminiscent of the polymerase domains of the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, and bacteriophage T7 RNA polymerase. The ?amino-terminal 8-kilodalton domain is attached to the fingers subdomain by a '''flexible hinge'''. '''The two invariant ?aspartates found in all polymerase sequences and implicated in catalytic activity have the same geometric arrangement within structurally similar but topologically distinct palms, indicating that the polymerases have maintained, or possibly re-evolved, a ?common nucleotidyl transfer mechanism.''' The location of Mn2+ and ?deoxyadenosine triphosphate in pol beta confirms the role of the invariant aspartates in metal ion and deoxynucleoside triphosphate binding.

8. (good) Crystal Structure of a pol Family Replication DNA Polymerase from Bacteriophage RB69

Wang, J., Sattar, A. K. M. A., Wang, C. C., Karam, J. D., Konigsberg, W. H., and Steitz, T. A. (1997) Cell 89, 1087–1089

The 2.8 A resolution crystal structure of the bacteriophage RB69 gp43, a member of the eukaryotic pol alpha family of replicative DNA polymerases, shares some similarities with other polymerases but shows many differences. Although its ?palm domain has the same topology as other polymerases, except rat DNA polymerase beta, one of the three ?carboxylates required for ?nucleotidyl transfer is located on a different beta strand. The structures of the fingers and thumb domains are unrelated to all other known polymerase structures. The editing 3'-5' exonuclease domain of ?gp43 is homologous to that of E. coli DNA polymerase I but lies on the opposite side of the ?polymerase active site. An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukaryotic DNA polymerases.

http://www.sciencedirect.com.ezproxy.lib.utexas.edu/science?ob=ArticleURL&udi=B6WSN-41XW7YR-F&user=108429&coverDate=06%2F27%2F1997&rdoc=1&fmt=&orig=search&sort=d&view=c&acct=C000059713&version=1&urlVersion=0&userid=108429&md5=97d0d743cb20ee0750b65709a5572075

The structure of this replication enzyme is organized into five domains that are arranged around a central hole (Figure 2). The polymerase has an overall disk-like shape with three clefts or grooves emanating from the central hole, which contains the polymerase active site. The structure of a complex between single-stranded DNA and the exonuclease domain of gp43 (see below) suggests that one cleft (which lies between the 3′–5′ exonuclease and polymerase active sites) binds the primer strand in the editing mode. Comparison of the structure of gp43 with that of Taq polymerase complexed with DNA ([22]) suggests that a second cleft binds the duplex DNA product, while the third cleft may bind the single-stranded portion of the template strand. There are two striking protrusions from this disk-like structure that may be important in organizing the replisome (see below). One is a hydrophobic stretch of a dozen residues at the C-terminus that extends outward from the putative product-binding cleft. The other is a largely antiparallel coiled-coil extension of the fingers domain located at the “back” of the molecule next to the central hole ( Figure 2B and Figure 2C).

• 1. S.H. Eom, J. Wang and T.A. Steitz, The structure of Taq polymerase with DNA at the polymerase active site. Nature 382 (1996), pp. 278–281. Full Text via CrossRef

The structures of the E. coli Klenow fragment (KF) and Thermus aquaticus DNA polymerase (Taq pol) from the pol I family ([49 and 36]) and that of the HIV reverse transcriptase (RT) ( [38]) and their DNA complexes ( [2, 28 and 22]) show a U-shaped polymerase domain geometry that has been likened to the shape of a hand with “thumb,” “palm,” and “fingers” subdomains ( [49 and 38]). The thumb interacts across the minor groove of product duplex DNA, while the palm contains the polymerase catalytic site; the fingers interact with the template strand and perhaps with the deoxynucleoside triphosphate. While the structure of mammalian DNA polymerase β (pol β) exhibits an analogous hand-like shape ( [17, 52 and 61]), it is not structurally homologous to pol I, RT, or T7 RNA polymerase (T7 RNAP) ( [66]) but rather belongs to another family of nucleotidyl transferases ( [26 and 73]).

9. DNA Polymerases: Structural Diversity and Common Mechanisms

Steitz, T. A. (1999) J. Biol. Chem. 274, 17395–17398

http://www.jbc.org.ezproxy.lib.utexas.edu/cgi/content/full/274/25/17395

Possibly the earliest enzymatic activity to appear in evolution was that of the polynucleotide polymerases, the ability to replicate the genome accurately being a prerequisite for evolution itself. Thus, one might anticipate that the mechanism by which all polymerases work would be both simple and universal. Further, these enzymatic scribes must faithfully copy the sequences of the genome into daughter nucleic acid or the information contained within would be lost; thus some mechanism of assuring fidelity is required. Finally, all classes of polynucleotide polymerases must be able to translocate along the template being copied as synthesis proceeds. The crystal structures of numerous DNA polymerases from different families suggest that they all utilize an identical two-metalioncatalyzed polymerase mechanism but differ extensively in many of their structural features.

Independent of their detailed domain structures, all polymerases whose structures are known presently appear to share a common overall architectural feature. They have a shape that can be compared with that of a right hand and have been described as consisting of "thumb," "palm," and "fingers" domains (15). '''The function of the ?palm domain appears to be catalysis of the ?phosphoryl transfer reaction whereas that of the ?fingers domain includes important interactions with the incoming ?nucleoside triphosphate as well as the ?template base to which it is paired. The thumb on the other hand may play a role in ''positioning'' the duplex DNA and in ''processivity'' and ''translocation''.''' Although the palm domain appears to be homologous among the pol I, pol , and RT families, '''the fingers and thumb domains are different in all four of these families''' for which structures are known to date (16).

Here the functional and structural similarities and differences among the polymerases of known structure are explored. Of particular interest are the role of editing in the '''fidelity of copying''', the ?common enzymatic mechanism of polymerases, and the manners in which different domain structures function in the polymerase reaction in analogous ways.

10. WHY DOES THIS HAVE NO NAME?

Kunkel, T. A., and Wilson, S. H. (1998) Nat. Struct. Biol. 5, 95–99

Recent crystal structures of DNA polymerases provide new insights into conformational dynamics and DNA minor groove interactions that are critical for efficient and accurate DNA synthesis.

Thomas A. Kunkel and Samuel H. Wilson are in the Laboratory of Structural Biology, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709, USA email: kunkel@niehs.nih.gov

http://www.nature.com.ezproxy.lib.utexas.edu/nsmb/journal/v5/n2/full/nsb0298-95.html

Critical to the stable transmission of genetic information is the proper functioning of DNA polymerases. These enzymes appear to catalyze a ?common nucleotidyl transfer reaction and their polymerase domains share common structural features. Nevertheless, as required for participation in a wide variety of DNA transactions that includes replication, recombination and several distinct DNA repair processes, DNA polymerases interact with different accessory proteins and DNA substrates and have a host of specialized properties and ?ancillary enzymatic activities. Our appreciation of both the similarities and differences among DNA polymerases has grown substantially in the past few months due to exciting new structural information on three of these enzymes (Table 1).

Two structures have been described of DNA polymerase (pol ) bound to the substrates it uses and one more to the product it generates during base excision repair of DNA damage in vivo1. The structure of bacteriophage T7 DNA polymerase (T7 pol) bound to a template-primer with a nucleoside triphos-phate in the polymerase active site has also just recently been described2, providing the first structure of a ternary complex for a member of the large Pol I family of DNA polymerases. The T7 pol was also complexed with an accessory protein (?thioredoxin) which confers high processivity to the polymerase, '''providing the first atomic level view of a simple and efficient multiprotein DNA replication machine'''. At the same time, three high resolution structures of ?thermostable Bacillus stearothermophilus DNA polymerase (?BF pol) have also been described3. Remarkably, this Pol I family enzyme is able to polymerize DNA in the crystal, thus establishing the orientation of the polymerase on the template-primer during synthesis. These structures provide rich detail on how DNA polymerases bind to and reshape their substrates and undergo ?conformational changes themselves in order to catalyze the processive and accurate synthesis needed to stably replicate and repair genomes.

?Conformational dynamics The recent structural studies indicate that the ?polymerase active site is assembled upon binding of the template-primer and the dNTP through a number of remarkable conformational changes in both the polymerase and the nucleic acid. '''Without these changes, a catalytically-competent active site simply does not exist.''' The structure of the ?ternary complex of pol bound to a single-nucleotide 16 base pair gapped substrate reveals a '''90° kink in the DNA''', located at the 5'-phosphodiester linkage between the ?template residue in the polymerase active site and the downstream single-stranded template base. This sharp bend exposes the base pair on the downstream side of the active site, allowing that template-strand base to stack with ?His 34 in helix B of the N-terminal 8,000 ?Mr domain of pol (Fig. la). '''The nucleic acid does not pass through a cleft in the protein but instead traverses the surface.''' The 5'-terminal base of the single nucleotide gap is bound to the ?apurinic/?apyrimidinic (AP) ?lyase active site (used for ?base excision repair) in the 8,000 Mr domain, and is thus quite distal to the 3'-OH of the primer terminus. '''This amazing structure leads to the interesting issue of how three enzymatic activities are coordinated in base excision repair: AP lyase activity to remove the sugar-phosphate, polymerization to fill the gap and ligation to complete base excision repair.'''

• (1) AP lyase activity to remove the sugar-phosphate
• (2) polymerization to fill the gap
• (3) ligation to complete base excision repair

Fig. 1 Stacking of pol and T7 pol histidine residues with template-strand bases downstream of the active site. Results are for the a, ternary pol and b, T7 pol complexes2. In both complexes, the incoming nucleotide (yellow) is paired with a template base (red) in the active site, while the next template-strand base (labeled n+1) is stacked with a histidine (blue). For clarity, only the 8,000 Mr (pol ) or fingers (T7 pol) subdomains are shown (white). The 5'-termini of the sugar-phosphate backbones are shown for the template strand (red) and the primer strand (lavender). In (a), the DNA complementary to the 5'-end of the template strand, which forms the single-nucleotide gap, is shown in green. The figure for pol is reproduced from ref. 2, with permission.

Fig. 2 Binding pockets for the nascent base pair in ternary complexes. Results are for the a, ternary pol 1 and b, T7 pol complexes2. The perspective is of the DNA major groove and illustrates the solvent exposure of the nascent base pair. The van der Waals surface of the templating base is in red and that of the incoming ddNTP is in yellow. Both protein (blue) and DNA (green) contribute to the binding pocket. The figure for pol is reproduced from ref. 26, with permission. This figure was prepared using the program GRASP27. The kinked configuration of the template strand provides access for specific amino-acid side chains of pol to interact with the template base pair located in the polymerase active site binding pocket (Fig. 2a). Comparing the structures of the pol complexes with and without the correct incoming ?ddCTP (Table 1) ''''reveals a major conformational change wherein the C-terminal subdomain of the polymerase rotates around the axis of -helix M to produce main chain movements of up to 11 Å '''. This substrate-induced subdomain closure around the DNA is accompanied by numerous smaller (1−2 Å;) conformational changes (Table 2, Figs 5, 6 in ref. 1, and pol movies on the internet at http://www-chem.ucsd.edu/Faculty/ Kraut/bpol.html ) which '''collectively generate an active site that is poised for catalysis.''' The polymerase components of the ?binding pocket for the ?nascent base pair (Fig. 2a) include residues ?Arg 283 and ?Lys 280, '''which interact with the template base''', and residues ?Asn 279 and ?Asp 276, '''which interact with the incoming dNTP'''.

hack Arg 283 & Lys 280 re: "ghost template strand" (virtualization)

'''The terminal base pair of the primer stem forms the other side of this pocket, which has a geometry that precisely accommodates a correct base pair.''' The recent structure of the T7 DNA pol ternary complex shares much in common with pol . The '''template-primer that is bound to T7 pol is also bent''', but in this case two bends are observed. One is in the duplex primer stem, where numerous interactions with the thumb subdomain occur (Fig. 3b). This bend is reminiscent of the 40−45° bend observed in duplex DNA bound to HIV-1 reverse transcriptase4. A second bend is seen where interactions occur between ?single-stranded template nucleotides and amino acids in the fingers subdomain. '''Together, the two bends result in a template-primer that is shaped like the letter S.''' The template nucleotide immediately downstream of the active site is flipped out and stacks with His 607 (Fig. Ifr), a situation reminiscent of the template base Atacking interaction with His 34 in the pol ternary complex (Fig. la). '''In this flipped out position, the next template base cannot form a Watson-Crick base pair with an incoming dNTP, suggesting that a conformational change occurs during ?translocation.'''

'''The single-stranded template nucleotides do not pass through a cleft formed by the thumb and fingers sub-domains, but rather lie on the surface of the protein.'''

Comparison of the structure of the T7 pol ternary complex to unliganded or binary complexes of ?Klenow fragment and ?Taq DNA pols (both also Pol I family enzymes) '''suggests that the fingers of T7 pol rotate 41° toward the template-primer (see Fig. 5a in ref. 2).''' '''As with pol , this major closure of a polymerase subdomain actually assembles the active site and the binding pocket for the nascent base pair (Fig. 2b).''' Again, this ?binding pocket is '''defined by both nucleic acid and protein, that is, the correct 3'-terminal base pair forms one side of the pocket and specific amino acids in the O helix form the other side.''' The resulting slot provides a '''tight fit (Fig. 2) for incorporation of the correct nucleotide.''' The recently described structures of BF pol−template-primer complexes (3) are the highest resolution structures yet reported for any DNA polymerase. The second and third BF pol−DNA structures listed in Table 1 demonstrate the incorporation of either one or two correct nucleotides, respectively, in the crystal. '''This amazing result establishes the orientation of this enzyme on the template-primer during binding, catalysis and translocation.''' The binary BF pol−DNA complexes lack bound dNTP and therefore represent a different step in the ?polymerase reaction cycle than the pol and T7 pol ternary complex structures. Nonetheless, these binary complexes share several features with the pol p and T7 pol ternary complexes. The terminal base pair is contained within a binding pocket (see Fig. 3 in ref. 3) that precisely accommodates a correct base pair and whose geometry is not optimal for binding a mismatched base pair. '''This pocket is defined by the correct penultimate base pair and by amino acid residues that hydrogen bond, stack, and/or provide van der Waals interactions with the terminal base pair.''' Among these is ?Tyr 714, which stacks with the template base of the terminal base pair. Tyr 714 is thus in the position one might have predicted for the first single stranded template base, which is instead flipped out due to a 90° bend in the template strand. '''As noted by Kiefer et al.3, a conformational change involving significant movement of the O helix in the fingers subdomain would be required to allow the next template base to correctly pair with an incoming dNTP.''' '''Thus, it is possible that the BF pol−DNA binary complex represents the structure of the polymerase following incorporation, but prior to, or in the process of, translocation.''' Fig. 3 Interactions of pol and T7 pol with the minor groove of duplex DNA. Results are for the a, ternary pol 6 and b, T7 pol complexes2. The left and right halves of each panel illustrate opposite sides of the complexes. For orientation, the catalytic residues of the polymerase active sites are in red and the 5'-ends of the primer strands are also indicated. Dark blue color represents the surface for interaction between the polymerase and the DNA, where distances between the DNA and protein molecular surfaces are less than 1.4 Å . This figure was prepared using the program ?GRASP27.

'''Active site mechanisms''' The ternary complex structures of pol and T7 pol have ddNMP at the 3' end of the primer, which was employed to freeze the reaction and capture the ternary complexes prior to catalysis. Thus, the primer 3'-hydroxyl oxygen is missing in these structures. Nevertheless, a modeled ?3'-hydroxyl oxygen is perfectly positioned in both structures to conduct an ?in-line nucleophilic attack on the -phosphorous of the incoming nucleotide. The ?phosphoryl transfer chemistry suggested by the ternary complexes of these two very different DNA polymerases is remarkably similar and shares several key features. Both enzymes have a network of '''stabilizing interactions around the ?triphosphate moiety of the incoming nucleotide.''' Both enzymes employ two metal ions that are coordinated with water molecules and ?strictly conserved carboxylates (?Asp 190, ?Asp 192 and ?Asp 256 in pol ; ?Asp 475 and ?Asp 654 in T7 pol). The metals are in optimal geometric positions to promote catalysis by the ?two metal ion mechanism (5). One metal ion activates the attacking anion, the 3'-hydroxyl oxygen, and also stabilizes the ?pentacovalent bipyrimidal intermediate of the transition state by coordinating with a ?nonbridging -phosphorous oxygen. The other metal ion is positioned to coordinate with ?nonbridging triphosphate oxygens as an ,,-tri-dentate; this metal ion also stabilizes the transition state by coordinating with the nonbridging -phosphate oxygen. In both enzymes, the negative charge developing on the -phosphate oxygen of the leaving group is not stabilized by a metal ion or by an enzyme group. Binding in the minor groove A feature common to the three polymerases listed in Table 1 (and to earlier pol−DNA complexes as well6−9) is that they bind to nucleic acids through interactions in the minor groove (Fig. 3). Polymerase interactions are not observed with the major groove (Fig. 3). The minor groove interactions are largely sequence independent, as required of DNA polymerases that replicate templates of widely differing sequence. Nevertheless, the polymerases vary with respect to the nature and number of these nucleic acid interactions. In the pol structures, most of the contacts between the 31,000 Mr polymerase domain and the template-primer are in or near the active site; little interaction is observed with the upstream duplex (Fig. 3a, Table 2). In contrast, the T7 pol ternary complex structure reveals interactions not only in and near the active site(Figs Ibj 2b)y but also numerous direct and water-mediated interactions of fingers, palm and thumb residues with the phosphate backbone upstream of the polymerase active site (Fig. 3b, also see Fig. 2 in ref. 2). Extensive contacts have been observed in a binary complex of Taq pol bound to blunt-ended duplex DNA7 and in a binary complex of DNA bound to Klenow fragment in an editing mode8 (see Fig. 2c in ref. 7). This picture of minor groove interactions is reminiscent of structure-function studies9 suggesting that HIV-1 RT binds to nucleic acid through a row of adjacent amino acids that protrude into the minor groove of the duplex 2 to 6 base pairs upstream of the active site. Again, RT interactions with the major groove are not apparent. In the new T7 pol (Fig. 3b) and BF pol structures, two or four base pairs respectively, adjacent to the 3'-primer terminus are significantly underwound such that the minor grooves are wider and shallower than B-form DNA. In contrast, duplex DNA that is not contacted by enzyme is B-form. DNA that is more like A-form near the polymerase active site has also been reported for HIV-1 RT bound to an 18−19−mer oligonucleotide4, for Taq pol bound to a blunt-ended duplex7 and for a ternary complex of pol bound to correct ddCTP and a 7−11-mer oligonucleotide6 (Fig. 3a). Concomitant with widening and flattening of the minor groove, numerous hydrogen bonds are observed between T7 and BF pols and the O2 atoms of pyrimidines and the N3 atoms of purines in the minor groove (Table 2). These contacts, which extend for 4 to 5 base pairs upstream of the active sites, have important functional implications. Table 1 Functions and recent structures of three DMA polymerases T7 BF1 Molecular mass (MT) 39,000 80,000(+12,000)2 68,000 Pol family Pol Poll Poll Primary function base excision repair replication unknown 3'5' exo no (but has AP lyase) yes3 no Processivity domain 8,000 MT domain thioredoxin — DNA substrate 1 -base gap4 1 -base gap4 product5 22/2S6 9/14 10/14 11/13 dNTP none ddCTP none ddGTP none none7 none8 Resolution (Å) 2.4 2.2 2.6 2.2 1.8 1.9 1.8 Active site metals 2Mg++ 2 Mg++ 1 Mg++ Stacking with template strand base His 34 His 607 Tyr714 DNA unwound near active site No9 Yes Yes 1Bacillus stearothermophilus DNA polymerase lacking the N-terminal 5'3' exonuclease, analogous to the Klenow fragment of E. coli DNA poly-merase I. 2The molecular weight of E. coli thioredoxin. 3Although the wild-type polymerase has 3'5' exonuclease activity, the structure is for an exonuclease-deficient T7DNA polymerase lacking six amino acids. 4The substrate contains a template guanine in the gap, formed by hybridization of a 16-mer with oligonucleotides of five and ten bases. 5The sequence is not the same as for the one base gap. Instead of a gap, it contains a primer-strand G opposite the template C, and thus no incoming dNTP. Therefore, this is the structure of a nicked 'product' complex. 6The numbers indicate the length of the DNA strand. The primer strand is listed first, then the template strand, with the difference being the length of the remaining single-stranded template. 7BF−DNA co-crystals were incubated with the ddTTP, which was subsequently incorporated as a correct nucleotide. 8The starting DNA contained nine base pairs of duplex DNA and two identical template overhangs of four single-stranded nucleotides. The ?BF−DNA co-crystal was incubated with dATP, with subsequent incorporation of two correct dAMP residues. 9Although the DNA is not unwound near the active site for the complexes described in ref. 1, it is more A-form like near the active site for the ternary complex with a 7−11-mer described in ref. 6. Processivity Consideration of the new structural information suggests several distinct ways whereby DNA polymerases achieve pro-cessive synthesis. On one extreme, pol dissociates from long single-stranded templates following each incorporation event. As the smallest of the DNA polymerases, its polymerase domain has limited contacts with the duplex primer stem (Fig. 30, Table 2). However, pol contains a unique 8,000 Mr N-terminal domain harboring the AP lyase active site that participates in base excision repair. This site can bind tightly to the downstream 5'-phosphate group in short gapped substrates, thus conferring on this DNA polymerase the ability to conduct processive synthesis to fill short gaps10. Pol I family polymerases such as T7 pol and BF pol have somewhat higher proces-sivity (10−100 incorporations per cycle11'12), consistent with more numerous interactions of their polymerase domains with template-primer, as discussed above. Synthesis by HIV-1 RT is also processive, but current information9 suggests that the extensive hydrophobic interactions of that polymerase with the minor groove of the duplex DNA may differ from those of the predominantly ionic interactions of Pol I family enzymes with duplex DNA. The structure of 17 pol2 further suggests how replication machines may be organized to achieve much higher proces-sivity. T7 pol complexed with E. coli thioredoxin is the simplest processive replication machine. In the absence of thioredoxin, T7 pol has moderate proces-sivity, but in its presence the polymerase copies thousands of nucleotides without dissociation12, a property which facilitates the efficient replication of large genomes. The T7 pol structure reveals that thioredoxin binds to a flexible, extended loop in the thumb subdomain of T7 pol that is not present in other Pol I family enzymes and is rich in glycine, proline and lysine residues. The authors hypothesize that this thioredoxin-bound loop could swing across the DNA binding groove to encircle the template-primer exiting the polymerase. This could enhance processivity either by providing electrostatic interactions with the DNA or by forming an enclosure over the DNA that functions like a sliding clamp13. The strategy of using a topologically-constrained sliding clamp to tether DNA polymerase to a template-primer in order to achieve processive synthesis is common to prokaryotic and eukaryotic repli-somes14. These include bacteriophage T4, where gp45 protein is the sliding clamp for highly processive T4 DNA replication. Insight into how this replisome may be organized comes from the exciting description of the crystal structure of RB69 DNA polymerase15, a homologue of T4 DNA polymerase. This is the first structural description of a member of the Pol family of DNA polymerases which includes the enzymes that replicate eukaryotic genomes. RB69 pol has three clefts and five subdomains, including fingers and thumb subdomains that are different from those of polymerases in other families. Based on the structure of the unliganded RB69 polymerase, modeling and biochemical observations, Wang et al.15 suggest that the processivity clamp interacts with a site in the C-terminal tail of the polymerase. Future studies of how clamp proteins partner with other proteins is critical, since clamp proteins participate in a wide variety of nucleic acid transactions. Polymerase fidelity Two structural features are particularly relevant to the fidelity of DNA synthesis. The most obvious is the geometry of the binding pocket for the nascent (Fig. 2) or terminal (BF pol) base pair. In the three recent articles1−3, the authors emphasize that this pocket tightly accommodates a canonical Watson-Crick base pair but would not accommodate incorrect base pairs due to steric constraints. That polymerase fidelity depends primarily on geometric selection based on the shape of the nascent base pair is supported by a variety of biochemical observations (see ref. 16 for a recent review). Relevant recent evidence includes the high selectivity of Klenow fragment pol for a base analog that lacks base−base hydrogen bonding potential yet has the shape of a correct base pair17, and the lower base selectivity of pol P18'19 and Klenow fragment polymer ases20'21 that contain changes in amino acid residues that may define the normal binding pocket. Table 2 Sequence-independent hydrogen-bonding to nucleic acid1 bases at the O2 and N3 atoms in the minor groove1 Primer T7 BF Template T7 BF dNTP - - -(R615) TO +(R283) - +(Q797) P1 + (Y271) +(R429) + T1 + (Q615) + P2 + (H653) + T2 + P3 + (Q439) + T3 + (N436) + P4 + (K394) + T4 + (Q439) + Total 1 4 5 1 3 5 1Results are taken from references 1−3. T is for template strand bases; P is for primer strand bases. TO is the template base in the active sites for pol and T7 pol. The "TO" and "dNTP" interactions listed for BF pol correspond to those observed with the terminal base pair in the BF pol binary complex. T1 and P1 (etc.) are for the upstream bases in the duplex. In parentheses are listed the amino acid residues that H-bond to O2 and N3 atoms The second feature is the pattern of hydrogen bonding between the poly-merases and the O2 and N3 atoms of bases of the duplex DNA that is upstream of the active site (Table 2). These hydrogen bond acceptors lie in the minor groove in the same positions for all four correct base pairs but are in different positions for incorrect base pairs. Thus, polymerases may probe the positions of these atoms to test for correct base pair geometry. The existing structures of pol ternary complexes1'6 and the reduced base selectivity of R283 pol mutants18'19, suggest that such probing can occur within the active site itself. The structures of all three polymerases also reveal hydrogen bonds to the primer-terminal base pair (Table 2). Since this base pair comprises one face of the binding pocket, the ability to sense incorrect geometry at this position may explain why polymerases fail to efficiently incorporate correct bases when a mismatched or damaged base pair is present at the primer terminus. For polymerases that have associated exonuclease activities (for example, T7 pol, Table 1), proofreading contributes on average 100-fold to base substitution fidelity22. Proofreading efficiency critically depends on the balance between the rates of polymerization and excision23. The ability to sense correct geometry at both the primer terminus and at some distance upstream of the active site (Table 2) likely contributes to the editing efficiency, since mismatches even four base pairs upstream of the active site may be seen as incorrect, tripping the balance in favor of excision. With this concept in mind, it will be very interesting to know the contacts between duplex DNA and Pol a family polymerases, since the location of the 3' 5' exonuclease active site relative to the polymerase active site is different in the structure of unliganded RB69 pol15 and that seen in the Pol I family enzymes. It is also noteworthy that proofreading efficiency can vary over a wide range and can be minimal, for example, for frameshift errors arising in homopolymeric runs of 6−8 base pairs24. The misaligned intermediates for such errors can have an extra base located in the primer stem at distances beyond that which a polymerase can probe for the correct positioning of O2 and N3 atoms (Table 2), thus favoring polymerization over excision. Differential hydrogen bonding with the primer stem may explain the sometimes remarkable differences in the frameshift fidelity of proofreading-deficient DNA polymerases. For example, for single base deletions at a particular template TTTT run, pol is 1000-fold less accurate than is exonuclease-deficient Klenow fragment pol25. Hydrogen bonding in the minor groove is also likely to be relevant to the outcome of encounters between different polymerases involved in replication, repair and recombination with abasic sites or bases (incoming dNTP or in the DNA) that have been damaged by chemical or physical agents. The fact that polymerases strongly influence the structure of the template-primer and that extensive substrate-induced conformational changes are required for polymerization implies that a full understanding of the consequences of an encounter of a polymerase with a damaged substrate will require information on the structure of ternary complexes. Acknowledgments We thank T. Ellenberger for generously providing the structural coordinates for T7 pol prior to publication of that study. We also thank W. Beard for preparation of the figures.

mediawiki reference barf

# Sawaya, M.R., Prasad, R., Wilson, S.H., Kraut, J. & Pelletier, H. ''Biochemistry'' '''36''', 11205−11215 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1021%2Fbi9703812 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9287163&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997XW73300018&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXlsV2nt7o%3D&pissn=1545-9993&pyear=1998&md5=31732d723e5de51ded3cab547ab84d6f ChemPort] |
# Doublié, S., Tabor, S., Long, A., Richardson, C.C. & Ellenberger, T. ''Nature'', in the press (1998).
# Kiefer, J.R., Mao, C, Braman, J.C. & Beese, L.S. ''Nature'', in the press (1998). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9440698&dopt=Abstract <span class="intlink">PubMed</span>] |
# Jacobo-Molina, A. ''et al'' ''Proc. Natl. Acad. Sci. USA'' '''90''', 6320−6324 (1993). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=7687065&dopt=Abstract <span class="intlink">PubMed</span>] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3sXltFahsrg%3D&pissn=1545-9993&pyear=1998&md5=fe3d1e1a40ee5f88cfe9677548b77a28 ChemPort] |
# Beese, L.S. & Steitz, T.A. ''EMBO J.'' ''' 10''', 25−33 (1991). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1989886&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1991EU78700004&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3MXhtFOlsb4%3D&pissn=1545-9993&pyear=1998&md5=6414bb98e4a1d7f2f21883c15666a4ce ChemPort] |
# Pelletier, H. Sawaya, M.R., Kumar, A., Wilson, S.H. & Kraut, J. ''Science'' '''264''', 1891−1903 (1991).
# Eom, S. H., Wang, J. & Steitz, T.A. ''Nature'' '''382''', 278−281 (1996). | [/doifinder/10.1038%2F382278a0 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8717047&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1996UX79000057&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK28XksVWhurY%3D&pissn=1545-9993&pyear=1998&md5=55899c2843249cd97761e0a8473b1eb5 ChemPort] |
# Beese, L.S., Derbyshire, V. & Steitz, T.A. ''Science'' '''260''', 352−355 (1993). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8469987&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1993KX80000038&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3sXisFShs7s%3D&pissn=1545-9993&pyear=1998&md5=a5fc038053428b4e6364da2c03df7397 ChemPort] |
# Bebenek, K. ''et al''. ''Nature Struct. Biol.'' '''4''', 194−197 (1997). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9164459&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997WM07100012&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXhslehurs%3D&pissn=1545-9993&pyear=1998&md5=a5081797c1b82836c5e042dbbe9256fe ChemPort] |
# Singhal, R.K. & Wilson, S.H. ''J. Biol. Chem.'' '''268''', 15906−15911 (1993). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8340415&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1993LN30500083&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3sXltlChsLc%3D&pissn=1545-9993&pyear=1998&md5=253812ccd27cdf655e25cae18daba6ba ChemPort] |
# Kiefer, J.R. ''et al''. ''Structure'' '''5''', 95−108 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1016%2FS0969-2126%2897%2900169-X Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9016716&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997WF20400011&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXhtFCkur4%3D&pissn=1545-9993&pyear=1998&md5=b241813b4dd88b29a99daad544b7bd65 ChemPort] |
# Tabor, S., Huber, H.E. & Richardson, C. C. ''J. Biol. Chem.'' '''262''', 16212−16223 (1987). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=3316214&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1987K966900060&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaL2sXmtFeltLg%3D&pissn=1545-9993&pyear=1998&md5=0dd275751c0454603edce83661b40a09 ChemPort] |
# Kong, X.-P., Onrust, R., O'Donnell, M. & Kuriyan, J. ''Cell'' '''69''', 425−437 (1992). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1016%2F0092-8674%2892%2990445-I Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1349852&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1992HT07800006&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK38XisFWjsL4%3D&pissn=1545-9993&pyear=1998&md5=8a712fb092a8308ee66629d588545b50 ChemPort] |
# Stillman, B. ''Cell'' '''78''', 725−728 (1994). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8087839&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1994PG29600001&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2cXmslejur0%3D&pissn=1545-9993&pyear=1998&md5=e15d85851eda963529398285d4e11a99 ChemPort] |
# Wang, J. ''et al''. ''Cell'' '''89''', 1087−1099 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1016%2FS0092-8674%2800%2980296-2 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9215631&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997XG83000013&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXkt1KlsLY%3D&pissn=1545-9993&pyear=1998&md5=d845861de8cc7030e50504a516fac769 ChemPort] |
# Goodman, M.F. ''Proc.Natl. Acad. Sci. USA'' '''94''', 10493−10495 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1073%2Fpnas.94.20.10493 Article] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXmtlemsL0%3D&pissn=1545-9993&pyear=1998&md5=368888a90287bd506a33d3901e86143f ChemPort] |
# Moran, S., Ren, R.X.-F. & Kool, E.T. ''Proc. Natl. Acad. Sci. USA'' '''94''', 10506−10511 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1073%2Fpnas.94.20.10506 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9380669&dopt=Abstract <span class="intlink">PubMed</span>] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXmtlemsbY%3D&pissn=1545-9993&pyear=1998&md5=7a555b35ffcb816a45e0fcd5bc9128e6 ChemPort] |
# Beard, W.A. ''et al''. ''J. Biol. Chem.'' '''271''', 12141−12144 (1996). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1074%2Fjbc.271.21.12141 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8647805&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1996UL66000007&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK28XjtFagu7o%3D&pissn=1545-9993&pyear=1998&md5=b4646b9bd86d956acd91f82cc56537b0 ChemPort] |
# Ahn, J., Wernburg, B.G. & Tsai, M. ''Biochemistry'' '''36''', 1100−1107 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1021%2Fbi961653o Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9033400&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997WG07200015&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXhtlamt7g%3D&pissn=1545-9993&pyear=1998&md5=ee7c5db61011dc2ece8bec3506023e6a ChemPort] |
# Carroll, S.S., Cowart, M. & Benkovic, S. ''J. Biochemistry'' '''30''', 804−813 (1991). | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3MXjvVeltA%3D%3D&pissn=1545-9993&pyear=1998&md5=98cc966c8d3ed60f6fbdb7ceccba7b44 ChemPort] |
# Bell, J.B., Eckert, K.A., Joyce, C.M. & Kunkel, T.A. ''J. Biol. Chem.'' '''272''', 7345−7351 (1997). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1074%2Fjbc.272.11.7345 Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=9054433&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1997WN14700072&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK2sXhvFGqs7g%3D&pissn=1545-9993&pyear=1998&md5=f67bfadc3831bb54341245418a1c0d56 ChemPort] |
# Kunkel, T.A. ''J. Biol. Chem.'' '''267''', 18251−18254 (1992). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1526964&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1992JN50200001&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK38XltVGhsbs%3D&pissn=1545-9993&pyear=1998&md5=ba8014bc76ef0c6eec080e0db189926a ChemPort] |
# Wong, I., Patel, S.S. & Johnson, K.A. ''Biochemistry'' '''30''', 526−537 (1991). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1846299&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1991ET38100030&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3MXpslahtQ%3D%3D&pissn=1545-9993&pyear=1998&md5=2b9e52052e01e0b45b4321149cacd718 ChemPort] |
# Kroutil, L.C., Register, K., Bebenek, K. & Kunkel, T.A. ''Biochemistry'' '''35''', 1046−1053 (1996). | [http://dx.doi.org.ezproxy.lib.utexas.edu/10.1021%2Fbi952178h Article] | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=8547240&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1996TR17800044&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK28Xis1Oitw%3D%3D&pissn=1545-9993&pyear=1998&md5=caa8e1b66c38f38bb461ac699c7b1b46 ChemPort] |
# Kunkel, T.A. ''Biochemistry'' '''29''', 8003−8011 (1990). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1702019&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1990DW78500001&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK3cXltFSjsro%3D&pissn=1545-9993&pyear=1998&md5=85569e3462b96af5590ca319ea986a8d ChemPort] |
# Beard, W.A. & Wilson, S.H. ''Chemistry & Biology'', in the press (1998).
# Nicholls, A., Sharp, K.A. & Honing, B. ''Proteins'' '''11''', 281−296 (1991). | [http://www.ncbi.nlm.nih.gov.ezproxy.lib.utexas.edu/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=1758883&dopt=Abstract <span class="intlink">PubMed</span>] | [http://links.isiglobalnet2.com.ezproxy.lib.utexas.edu/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=A1991GV03200006&DestApp=WOS_CPL
 ISI] | [http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DyaK38XhtVWgur4%3D&pissn=1545-9993&pyear=1998&md5=18992ca40777e35036d669e8d1b8e703 ChemPort] |

11. oink

http://arjournals.annualreviews.org.ezproxy.lib.utexas.edu/doi/abs/10.1146/annurev.biochem.69.1.497

Kunkel, T. A., and Bebenek, K. (2000) Annu. Rev. Biochem. 69, 497–529

DNA replication fidelity is a key determinant of genome stability and is central to the evolution of species and to the origins of human diseases. Here we review our current understanding of ?replication fidelity, with emphasis on structural and biochemical studies of DNA polymerases that provide new insights into the importance of ?hydrogen bonding, ?base pair geometry, and ?substrate-induced conformational changes to fidelity. '''These studies also reveal polymerase interactions with the ?DNA minor groove at and upstream of the active site that influence ?nucleotide selectivity, the ?efficiency of exonucleolytic proofreading, and the ?rate of forming errors via strand misalignments.''' We highlight common features that are relevant to the fidelity of any DNA synthesis reaction, and consider why fidelity varies depending on the enzymes, the error, and the local sequence environment.

12. buh?

Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891–1903

13. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Ã… resolution

Doublie, S., Tabor, S., Long, A. M., Richardson, C. C., and Ellenberger, T. (1998) Nature 391, 251–258

http://ezproxy.lib.utexas.edu/login?url=http://www.nature.com/nature/archive/index.html

Steitz

'''DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle''', while achieving error frequencies in the range of 10^-5 to 10^-6. Here we present a 2.2 Å crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer–template and a nucleoside triphosphate in the polymerase active site. '''The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a ?metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.'''

http://www.nature.com.ezproxy.lib.utexas.edu/nature/journal/v391/n6664/abs/391251a0.html

14. Structure of a Covalently Trapped Catalytic Complex of HIV-1 Reverse Transcriptase: Implications for Drug Resistance

Huang, H., Chopra, R., Verdine, G. L., and Harrison, S. C. (1998) Science 282, 1669 –1675

A combinatorial disulfide cross-linking strategy was used to prepare a stalled complex of human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase with a DNA template:primer and a deoxynucleoside triphosphate (dNTP), and the crystal structure of the complex was determined at a resolution of 3.2 angstroms. The presence of a dideoxynucleotide at the 3'-primer terminus allows capture of a state in which the substrates are poised for attack on the dNTP. Conformational changes that accompany formation of the catalytic complex produce distinct clusters of the residues that are altered in viruses resistant to nucleoside analog drugs. The positioning of these residues in the neighborhood of the dNTP helps to resolve some long-standing puzzles about the molecular basis of resistance. The resistance mutations are likely to influence binding or reactivity of the inhibitors, relative to normal dNTPs, and the clustering of the mutations correlates with the chemical structure of the drug.

http://www.sciencemag.org.ezproxy.lib.utexas.edu/cgi/content/abstract/282/5394/1669

15. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation

Li, Y., Korolev, S., and Waksman, G. (1998) EMBO J. 17, 7514 –7525 The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer/template DNA and dideoxycytidine triphosphate, and that of a binary complex of the same enzyme with a primer/template DNA, were determined to a resolution of 2.3, 2.3 and 2.5 Å, respectively. '''One ternary complex structure differs markedly from the two other structures by a large reorientation of the tip of the fingers domain.''' This structure, designated 'closed', '''represents the ternary polymerase complex caught in the act of incorporating a nucleotide.''' In the two other structures, the tip of the fingers domain is rotated outward by 46° ('open') in an orientation similar to that of the apo form of Klentaq1. '''These structures provide the first direct evidence in DNA polymerase I enzymes of a large conformational change responsible for assembling an active ternary complex.'''

http://www.nature.com.ezproxy.lib.utexas.edu/emboj/journal/v17/n24/abs/7591437a.html

more notes

• DNA polymerase
• RNA polymerase
• DNA-dependent DNA polymerase
• RNA-dependent DNA polymerase
• DNA-dependent RNA polymerase
• RNA-dependent RNA polymerase
• telomerase
• reverse transcriptase (RNA-dependent DNA polymerase)
• nucleotidyl transferase https://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Nucleotidyltransferases

DNA-directed vs DNA-dependent ??

• Polyribonucleotide Nucleotidyltransferase (PNPASE) https://en.wikipedia.org/wiki/Polynucleotide_phosphorylase

• DNA-directed DNA polymerase

• DNA nucleotidylexotransferase (same as terminal deoxynucleotidyl transferase ??)

• templateless polymerases if any

also check

• recombinase
• CRISPR stuff
• helicase
• replicase
• DNA ligase
• RNA ligase
• RNA ligase ribozyme
• RNA helicase
• RNA replicase

terminal deoxynucleotide transferase

terminal deoxyribonucleotidyltransferase terminal transferase TdT addase activity

https://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase

• telomerase reverse transcriptase (TERT) https://en.wikipedia.org/wiki/Telomerase_reverse_transcriptase ** has an RNA component that encodes the template for addition

Large components

• Siderophore biosynthesis non-ribosomal peptide synthetase module http://www.uniprot.org/uniprot/A0A108U5E5

• Polymerase/replicase http://www.uniprot.org/uniprot/J7I0T0 -- RNA-directed RNA polymerase activity, RdRp catalytic peptide domain

Taq polymerase http://www.uniprot.org/uniprot/P19821

http://www.ebi.ac.uk/interpro/entry/IPR019760

""" DNA polymerases (EC:2.7.7.7) can be classified, on the basis of sequence similarity [PMID: 3479792, PMID: 2196557], into at least four different groups: A, B, C and X. Members of family X are small (about 40 kDa) compared with other polymerases and encompass two distinct polymerase enzymes that have similar functionality: vertebrate polymerase beta (same as yeast pol 4), and terminal deoxynucleotidyl-transferase (TdT) (EC:2.7.7.31). The former functions in DNA repair, while the latter terminally adds single nucleotides to polydeoxynucleotide chains. Both enzymes catalyse addition of nucleotides in a distributive manner, i.e. they dissociate from the template-primer after addition of each nucleotide. DNA-polymerases show a degree of structural similarity with RNA-polymerases.

Five regions of similarity are found in all the polymerases of this entry. The signature of this entry is to the conserved region, known as 'motif B' [PMID: 2196557]; motif B is located in a domain which, in E. coli polA, has been shown to bind deoxynucleotide triphosphate substrates; it contains a conserved tyrosine which has been shown, by photo-affinity labelling, to be in the active site; a conserved lysine, also part of this motif, can be chemically labelled, using pyridoxal phosphate. """

Catalytic domain matching

• RdRp catalytic domain matching http://www.uniprot.org/saas/SAAS00519020 view all proteins annotated by this rule http://www.uniprot.org/uniprot/?query=source:SAAS00519020

• • "Contains RdRp catalytic domain"

• DdDp catalytic domain matching

• RNA-directed RNA polymerase catalytic domain profiles http://prosite.expasy.org/PDOC50507

• Reverse transcriptase (RT) catalytic domain profile http://prosite.expasy.org/PDOC50878

• https://en.wikipedia.org/wiki/Reverse_transcriptase

• https://en.wikipedia.org/wiki/Telomerase_reverse_transcriptase
• https://en.wikipedia.org/wiki/DNA_polymerase
• https://en.wikipedia.org/wiki/RNA-dependent_RNA_polymerase
• https://en.wikipedia.org/wiki/RNA_polymerase
• https://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase

"Flaviviruses produce a polyprotein from the ssRNA genome. The polyprotein is cleaved to a number of products, one of which is NS5. Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. This RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases.[11]"

a template-independent polymerase like Terminal deoxynucleotidyl Transferase (TdT) or poly(A) polymerase

""" According to certain aspects, oligonucleotide sequences may be prepared using ink jet techniques known to those of skill in the art, electrochemical techniques known to those of skill in the art, microfluidic techniques known to those of skill in the art, photogenerated acids known to those of skill in the art, or photodeprotected monomers known to those of skill in the art. Such techniques have the advantage of making oligonucleotides at high speed, low cost, fewer toxic chemicals, enhanced portability and ability to interleave DNA biochemistry (e.g. modifications, polymerases, hybridization etc.) with de novo (digital or analog) synthesis. For example, spatially patterned light, either directly from camera optics or from Digital Micromirror Display devices (DMD), can be used with aqueous chemistry. See US2003/0228611. For example, a template-independent polymerase like Terminal deoxynucleotidyl Transferase (TdT) or poly(A) polymerase—alternatively, a template-dependent polymerase like Taq or Phi29 derivatives, can have their basic polymerase function, base-specificity or fidelity programmable by light by incorporating an azobenzene amino acid (see Hoppmann C, Schmieder P, Heinrich N, Beyermann M. (2011) Chembiochem. 12(17):2555-9. doi: 10.1002/cbic. 201100578. Epub 2011 October 13, Photoswitchable click amino acids: light control of conformation and bioactivity) into the active site of the polymerase or 5′43′ exonuclease domains (if present). """

... can have their basic polymerase function, base-specificity or fidelity programmable by light by incorporating an azobenzene amino acid (see Hoppmann C, Schmieder P, Heinrich N, Beyermann M. (2011) Chembiochem. 12(17):2555-9. doi: 10.1002/cbic. 201100578. Epub 2011 October 13, Photoswitchable click amino acids: light control of conformation and bioactivity) into the active site of the polymerase or 5′43′ exonuclease domains

see Hoppmann C, Schmieder P, Heinrich N, Beyermann M. (2011) Chembiochem. 12(17):2555-9. doi: 10.1002/cbic. 201100578. Epub 2011 October 13, Photoswitchable click amino acids: light control of conformation and bioactivity) into the active site of the polymerase or 5′43′ exonuclease domains

""" Light sensitive neurons (optogenetics) can trigger ion-sensitive polymerases (see Zamft B, Marblestone A, Kording K, Schmidt D, Martin-Alarcon D, Tyo K, Boyden E, Church GM (2012) Measuring Cation Dependent DNA Polymerase Fidelity Landscapes by Deep Sequencing. PLoS One, in press) or, for some applications, the ion flux patterns themselves can constitute the stored datasets. """

""" The nucleotide type incorporated can be determined by: a) the intersection of a light pulse coincident with a particular dNTP (or rNTP or other monomer class) present at that time point in a cyclic pattern of dNTP solutions. b) ‘caged’ (i.e. photo-activatable or photo-inactivatable) dNTPs, rNTPs or cations. c) base-specific, light-modulated steric or conformational selectivity (see Hoppmann C, Schmieder P, Heinrich N, Beyermann M. (2011) Chembiochem. 12(17):2555-9. doi: 10.1002/cbic.201100578. Epub 2011 October 13. Photoswitchable click amino acids: light control of conformation and bioactivity). Poly(A) polymerase is particularly useful since its specificity for ATp relative to other rNTPs is due to a conformational change which can be mimicked by a photo-sensitive amino acid linkage (like azobenzene, with or without crosslinking). """

Photoswitchable Click Amino Acids: Light Control of Conformation and Bioactivity http://onlinelibrary.wiley.com/doi/10.1002/cbic.201100578/abstract

"Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge (see figure). By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo."

Reversible photocontrol of biological systems by the incorporation of molecular photoswitches

Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells

SELEX

phage display

ribosome display

mRNA display

phage-assisted continuous evolution

TRAP display

liposome display

megavalent bead surface display

mRNA display selection

• azobenzene-matching aptamer, introduce azobenzene into the aqueous environment ?