New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis

Time-lapse X-ray crystallography allows visualization of intermediate structures during the DNA polymerase catalytic cycle. Employing time-lapse crystallography with human DNA polymerase β has recently allowed us to capture and solve novel intermediate structures that are not stable enough to be analyzed by traditional crystallography. The structures of these intermediates reveals exciting surprises about active site metal ions and enzyme conformational changes as the reaction proceeds from the ground state to product release. In this perspective, we provide an overview of recent advances in understanding the DNA polymerase nucleotidyl transferase reaction and highlight both the significance and mysteries of enzyme efficiency and specificity that remain to be solved.     

Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center

Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.     

Structure of human DNA polymerase iota and the mechanism of DNA synthesis

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX–XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.     

Multiple amino acid substitutions allow DNA polymerases to synthesize RNA

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 10(3)-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.     

Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site.     

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.     

New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions

The A-rule in mutagenesis was originally proposed to explain the preponderance of X-->T mutations observed for abasic sites and UV damaged sites. It was deduced that when a polymerase was faced with a non-instructional lesion, typified by an abasic site, it would preferentially incorporate an A. In the absence of any other compelling explanation, any lesion causing an X-->T mutation has often been classified as non-instructional to account for its apparent lack of instructional ability. The A-rule and the classification of lesions as non-instructional were formulated before the active sites of any polymerases or the mechanism by which they synthesized DNA were known. Since then, much structural and kinetic data on DNA polymerases has emerged to suggest mechanistic explanations for the A-rule and the instructive and non-instructive behavior of lesions such as cis-syn dimers. Polymerases involved in the replication of undamaged DNA have highly constrained active sites that evolved to only accommodate the templating base and the complementary nucleotide and as a result are relatively intolerant of modifications that alter the size and shape of the nascent base pair. On the other hand, DNA damage bypass polymerases have much more open and less constrained active sites, which are much more tolerant of modifications. An otherwise instructional lesion would become non-instructional if it were unable to fit into the active site, and thereby behave transiently like an abasic site, leading to the insertion of whichever nucleotide is favored by the polymerase, generally an A. In this review, what is known about the active sites and mechanisms of replicative and DNA damage bypass polymerases will be discussed with regard to the A-rule and non-instructive behavior of lesions, typified by dipyrimidine photoproducts.     

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.     

A single highly mutable catalytic site amino acid is critical for DNA polymerase fidelity

DNA polymerases contain active sites that are structurally superimposable and conserved in amino acid sequence. To probe the biochemical and structure-function relationship of DNA polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (Motif A; amino acids 605-617), and a fraction of all selected active Taq pol I (291 out of 8000) was tested for base pairing fidelity; seven unique mutants that efficiently misincorporate bases and/or extend mismatched bases were identified and sequenced. These mutants all contain substitutions of one specific amino acid, Ile-614, which forms part of the hydrophobic pocket that binds the base and ribose portions of the incoming nucleotide. Mutant Taq pol Is containing hydrophilic substitution I614K exhibit 10-fold lower base misincorporation fidelity, as well as a high propensity to extend mispairs. In addition, these low fidelity mutants containing hydrophilic substitution for Ile-614 can bypass damaged templates that include an abasic site and vinyl chloride adduct ethenoA. During polymerase chain reaction, Taq pol I mutant I614K exhibits an error rate that is >20-fold higher relative to the wild-type enzyme and efficiently catalyzes both transition and transversion errors. These studies have generated polymerase chain reaction-proficient mutant polymerases containing substitutions within the active site that confers low base pairing fidelity and a high error rate. Considering the structural and sequence conservation of Motif A, it is likely that a similar substitution will yield active low fidelity DNA polymerases that are mutagenic.     

Antimutator variants of DNA polymerases

Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino acid substitutions that lower intrinsic error rates. Here, we review these 'antimutagenic' changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient 'mutator' derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype.     

A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.     

Structures of mismatch replication errors observed in a DNA polymerase

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.     

Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.     

DNA structure and polymerase fidelity.

The accuracy of DNA replication results from both the intrinsic DNA polymerase fidelity and the DNA sequence. Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is less well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage including (CA)n and (A)n tracts in different intermolecular contexts reveals that, in the B-form, these sequences share common structural alterations which may explain the high rate of replication errors. In particular, a two-faced "Janus-like" structure with shifted base-pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy specifically suitable to mislead the polymerases. A model of the rat polymerase beta bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base-pairing, while at the same time escaping enzymatic mechanisms of error discrimination which scan for the correct geometry of the minor groove.In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study suggests that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The detailed comparison of the conformation of the nascent template-primer duplex in the available crystal structures of DNA polymerase-DNA complexes shows that polymerase beta, the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.     

NTP-entry routes in multi-subunit RNA polymerases

The recent elucidation of crystal structures for multi-subunit RNA polymerases immediately revealed a mystery: how do nucleotide triphosphate (NTP) substrates reach an active site that is buried deep within the enzyme? The prevailing view is that NTPs enter through an approximately 20A-long secondary channel between the active site and the enzyme surface. Recently, an alternative view has been advocated; namely, NTPs enter the active site pre-bound to the DNA template from the downstream DNA portion of the main channel of the enzyme.     

Binary system for selective photoaffinity labeling of base excision repair DNA polymerases

A system of photoaffinity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic fibroblast (MEF) cellular extract in the presence of base-substituted arylazido derivatives of dNTPs. This results in synthesis of a photoreactive long patch base excision repair (BER) intermediate. The arylazido photoreactive group is then activated through energy transfer from the pyrene group of a dNTP analog (Pyr-dUTP), following 365 nm UV light exposure. Pyr-dUTP binds to the active site of DNA polymerases, and the pyrene group, when excited by 365 nm UV light, activates the nearby photoreactive group in the BER intermediate resulting in crosslinking of DNA-bound DNA polymerases. Under these conditions, various DNA binding proteins that are unable to bind Pyr-dUTP are not crosslinked to DNA. DNA polymerase β is the predominant crosslinked protein observed in the MEF extract. In contrast, several other DNA binding proteins are labeled under conditions of direct UV light activation of the photoreactive group at 312 nm. This study illustrates use of a new method of selective labeling of DNA polymerases in a crude cellular extract.     

Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus.

A new group of error-prone DNA polymerases overcomes the blockage posed to normal DNA replication by damaged template bases, suggesting an active site with a loose, flexible pocket that accommodates aberrant DNA structures. We have determined a 2.8 A resolution crystal structure of the Sulfolobus solfataricus Dbh protein, a DNA translesion polymerase closely related to Escherichia coli DNA polymerase IV and human polymerase kappa. A high error rate is observed for the Dbh polymerase in a range of 10(-2)-10(-3) for all 12 base substitution mispairs. The crystal structure of Dbh reveals an overall architecture resembling other DNA polymerases but has unique features that are likely to contribute to error-prone synthesis, including -1 frameshifting mutations.