Lesion Bypass DNA Polymerases Research Articles

Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast

PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.

Replication of the Aristolochic Acid I Adenine Adduct (ALI-N6-A) by a Model Translesion Synthesis DNA Polymerase: Structural Insights on the Induction of Transversion Mutations from Molecular Dynamics Simulations.

Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N6-A and ALII-N6-A) adducts cause the "signature" A → T transversion mutations associated with AA genotoxicity. To provide the currently missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics simulations to examine different (i.e., preinsertion, insertion, and postextension) stages of replication past the most abundant AA adduct (ALI-N6-A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that, before dNTP incorporation (i.e., preinsertion step), ALI-N6-A adopts a nearly planar conformation at the N6-linkage and the ALI moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in a significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N6-A undergoes a small conformational change to introduce non-planarity at the N6-linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches and then result in A → T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, dTTP (non-mutagenic) incorporation will be favored. Molecular dynamics (MD) simulations on postextension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N6-A adducted DNA, with the exception of slight non-planarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N6-A and thereby rationalize the previously reported induction of A → T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.

Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity

DNA template damage can potentially block DNA replication. Cells have therefore developed different strategies to repair template lesions. Activation of the bacterial lesion bypass DNA polymerase V (Pol V) requires both the cleavage of the UmuD subunit to UmuD' and the acquisition of a monomer of activated RecA recombinase, forming Pol V Mut. Both of these events are mediated by the generation of RecA* via the formation of a RecA-ssDNA filament during the SOS response. Formation of RecA* is itself modulated by competition with the ssDNA-binding protein (SSB) for binding to ssDNA. Previous observations have demonstrated that RecA filament formation on SSB-coated DNA can be favored in the presence of the recombination mediator proteins RecF, RecO, and RecR. We show here using purified proteins that in the presence of SSB and RecA, a stable RecA-ssDNA filament is not formed, although sufficient RecA* is generated to support some activation of Pol V. The presence of RecFOR increased RecA* generation and allowed Pol V to synthesize longer DNA products and to elongate from an unpaired primer terminus opposite template damage, also without the generation of a stable RecA-ssDNA filament.

Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity.

The coding sequences of plant mitochondrial and chloroplast genomes present a lower mutation rate than the coding sequences of animal mitochondria. However, plant mitochondrial genomes frequently rearrange and present high mutation rates in their noncoding sequences. DNA replication in plant organelles is carried out by two DNA polymerases (DNAP) paralogs. In Arabidopsis thaliana at least one DNAP paralog (AtPolIA or AtPolIB) is necessary for plant viability, suggesting that both genes are partially redundant. To understand how AtPolIs replicate genomes that present low and high mutation rates, we measured their nucleotide incorporation for all 16-base pair combinations in vitro. AtPolIA presents an error rate of 7.26 × 10-5 , whereas AtPolIB has an error rate of 5.45 × 10-4 . Thus, AtPolIA and AtPolIB are 3.5 and 26-times less accurate than human mitochondrial DNAP γ. The 8-fold difference in fidelity between both AtPolIs results from a higher catalytic efficiency in AtPolIA. Both AtPolIs extend from mismatches and the fidelity of AtPolIs ranks between high fidelity and lesion bypass DNAPs. The different nucleotide incorporation fidelity between AtPolIs predicts a prevalent role of AtPolIA in DNA replication and AtPolIB in DNA repair. We hypothesize that in plant organelles, DNA mismatches generated during DNA replication are repaired via recombination-mediated or DNA mismatch repair mechanisms that selectively target the coding region and that the mismatches generated by AtPolIs may result in the frequent expansion and rearrangements present in plant mitochondrial genomes.

Plant organellar DNA polymerases are replicative and translesion DNA synthesis polymerases.

Genomes acquire lesions that can block the replication fork and some lesions must be bypassed to allow survival. The nuclear genome of flowering plants encodes two family-A DNA polymerases (DNAPs), the result of a duplication event, that are the sole DNAPs in plant organelles. These DNAPs, dubbed Plant Organellar Polymerases (POPs), resemble the Klenow fragment of bacterial DNAP I and are not related to metazoan and fungal mitochondrial DNAPs. Herein we report that replicative POPs from the plant model Arabidopsis thaliana (AtPolI) efficiently bypass one the most insidious DNA lesions, an apurinic/apyrimidinic (AP) site. AtPolIs accomplish lesion bypass with high catalytic efficiency during nucleotide insertion and extension. Lesion bypass depends on two unique polymerization domain insertions evolutionarily unrelated to the insertions responsible for lesion bypass by DNAP θ, an analogous lesion bypass polymerase. AtPolIs exhibit an insertion fidelity that ranks between the fidelity of replicative and lesion bypass DNAPs, moderate 3′-5′ exonuclease activity and strong strand-displacement. AtPolIs are the first known example of a family-A DNAP evolved to function in both DNA replication and lesion bypass. The lesion bypass capabilities of POPs may be required to prevent replication fork collapse in plant organelles.

Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

DNA–protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA–protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.

Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace, but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local side chain fluctuations in the apo, binary, and ternary states of Dpo4, we observed that the DNA binding interface and active site are dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation whereas the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.

Structural model of the Y-Family DNA polymerase V/RecA mutasome

To synthesize past DNA damaged by chemicals or radiation, cells have lesion bypass DNA polymerases (DNAPs), most of which are in the Y-Family. One class of Y-Family DNAPs includes DNAP η in eukaryotes and DNAP V in bacteria, which have low fidelity when replicating undamaged DNA. In Escherchia coli, DNAP V is carefully regulated to insure it is active for lesion bypass only, and one mode of regulation involves interaction of the polymerase subunit (UmuC) and two regulatory subunits (UmuD’) with a RecA-filament bound to ss-DNA. Taking a docking approach, ∼150,000 unique orientations involving UmuC, UmuD’ and RecA were evaluated to generate models, one of which was judged best able to rationalize the following published findings. (1) In the UmuD’2C/RecA-filament model, R64-UmuC interacts with S117-RecA, which is known to be at the UmuC/RecA interface. (2) At the model's UmuC/RecA interface, UmuC has three basic amino acids (K59/R63/R64) that anchor it to RecA. No other Y-Family DNAP has three basic amino acids clustered in this region, making it a plausible site for UmuC to form its unique interaction with RecA. (3) In the model, residues N32/N33/D34 of UmuC form a second interface with RecA, which is consistent with published findings. (4) Active UmuD’ is generated when 24 amino acids in the N-terminal tail of UmuD are proteolyzed, which occurs when UmuD2C binds the RecA-filament. When UmuD is included in an UmuD2C/RecA-filament model, plausible UmuD/RecA contacts guide the UmuD cleavage site (C24/G25) into the UmuD proteolysis active site (S60/K97). One contact involves E11-UmuD interacting with R243-RecA, where the latter is known to be important for UmuD cleavage. (5) The UmuD2C/RecA-filament model rationalizes published findings that at least some UmuD-to-UmuD’ cleavage occurs intermolecularly. (6) Active DNAP V is known to be the heterotetramer UmuD’2C/RecA, a model of which can be generated by a simple rearrangement of the RecA monomer at the 3′-end of the RecA-filament. The rearranged UmuD’2C/RecA model rationalizes published findings about UmuD’ residues in proximity to RecA. In summary, docking and molecular simulations are used to develop an UmuD’2C/RecA model, whose structure rationalizes much of the known properties of the active form of DNA polymerase V.

Kinetic Basis for the Differing Response to an Oxidative Lesion by a Replicative and a Lesion Bypass DNA Polymerase from Sulfolobus solfataricus

8-Oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), a major oxidative DNA lesion, exhibits ambiguous coding potential and can lead to genomic mutations. Tight control of 8-oxoG bypass during DNA replication is therefore extremely important in hyperthermophiles as the rate of oxidative damage to DNA is significantly increased at high temperatures. Here we employed pre-steady state kinetics to compare the kinetic responses to an 8-oxoG lesion of the main replicative and lesion bypass DNA polymerases of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon. Upon encountering 8-oxoG, PolB1, the replicative DNA polymerase, was completely stalled by the lesion, as its 3' → 5' exonuclease activity increased significantly and outcompeted its slowed polymerase activity at and near the lesion site. In contrast, our results show that Dpo4, the lone Y-family DNA polymerase in S. solfataricus, can faithfully and efficiently incorporate nucleotides opposite 8-oxoG and extend from an 8-oxoG:C base pair with a mechanism similar to that observed for the replication of undamaged DNA. Furthermore, we show that the stalling of PolB1 at the lesion site can be relieved by Dpo4. Finally, the 3' → 5' exonuclease activity of PolB1 was the highest when 8-oxoG was mispaired with an incorrect nucleotide and could therefore correct rare mistakes made by Dpo4 during 8-oxoG bypass. These results provide a kinetic basis for a potential polymerase switching mechanism during 8-oxoG bypass whereby Dpo4 can switch with the stalled PolB1 at the replication fork to bypass and extend the damaged DNA and then switch off of the DNA substrate to allow continued replication of undamaged DNA by the more faithful PolB1.

Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson–Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.

Architecture of Y‐Family DNA Polymerases Relevant to Translesion DNA Synthesis as Revealed in Structural and Molecular Modeling Studies

DNA adducts, which block replicative DNA polymerases (DNAPs), are often bypassed by lesion-bypass DNAPs, which are mostly in the Y-Family. Y-Family DNAPs can do non-mutagenic or mutagenic dNTP insertion, and understanding this difference is important, because mutations transform normal into tumorigenic cells. Y-Family DNAP architecture that dictates mechanism, as revealed in structural and modeling studies, is considered. Steps from adduct blockage of replicative DNAPs, to bypass by a lesion-bypass DNAP, to resumption of synthesis by a replicative DNAP are described. Catalytic steps and protein conformational changes are considered. One adduct is analyzed in greater detail: the major benzo[a]pyrene adduct (B[a]P-N2-dG), which is bypassed non-mutagenically (dCTP insertion) by Y-family DNAPs in the IV/κ-class and mutagenically (dATP insertion) by V/η-class Y-Family DNAPs. Important architectural differences between IV/κ-class versus V/η-class DNAPs are discussed, including insights gained by analyzing ~400 sequences each for bacterial DNAPs IV and V, along with sequences from eukaryotic DNAPs kappa, eta and iota. The little finger domains of Y-Family DNAPs do not show sequence conservation; however, their structures are remarkably similar due to the presence of a core of hydrophobic amino acids, whose exact identity is less important than the hydrophobic amino acid spacing.

Y-Family DNA polymerases may use two different dNTP shapes for insertion: A hypothesis and its implications

Chemicals and radiation can damage DNA leading to the formation of adducts/lesions, which – if not removed by DNA repair pathways – usually block replicative DNA polymerases (DNAPs). To overcome such potentially lethal blockage, cells have lesion bypass DNAPs, which are often in the Y-Family and include several classes. One class includes human DNAP κ and E. coli DNAP IV, and they insert dCTP in the non-mutagenic pathway opposite [+ta]-B[a]P-N 2-dG, which is the major adduct formed by the environmental carcinogen benzo[a]pyrene. Another class includes hDNAP η and ecDNAP V, and they insert dATP opposite [+ta]-B[a]P-N 2-dG in the dominant G → T mutagenic pathway. Herein we develop a hypothesis for why the IV/κ-class preferentially does cellular dCTP insertion. On the minor groove side of the active site, Y-Family DNAPs have a cleft/hole that can be analyzed based on an analogy to a “chimney.” Our models of DNAP IV show a large chimney opening from which the pyrene of [+ta]-B[a]P-N 2-dG can protrude, which allows canonical adduct-dG:dCTP pairing. In contrast, our models of DNAP V have small chimney openings that forces adduct-dG downward in the active site such that canonical adduct-dG:dCTP pairing is not possible. Based on X-ray structures, sequence alignment and our modeled structures of Y-Family DNAPs, chimney opening size seems primarily controlled by one amino acid (“flue-handle”), which dictates whether nearby amino acids (“flue”) plug the chimney or not. Based on this analysis, a correlation is apparent: the flue is closed in V/η-class DNAPs giving small chimney openings, while the flue is open for the IV/κ-class giving large chimney openings. Secondarily, a hypothesis is developed for why the V/η-class might preferentially do cellular dATP insertion opposite [+ta]-B[a]P-N 2-dG: the small chimney forces adduct-dG lower in the active site, possibly leading to catalysis using a non-canonical dNTP shape that permits syn-adenine:adduct-dG base pairing. In summary, a hypothesize is developed that the pyrene moiety of [+ta]-B[a]P-N 2-dG protrudes from the large chimney opening of DNAP IV, thus permitting canonical dCTP:adduct-dG pairing, while the small chimney opening of DNAP V forces [+ta]-B[a]P-N 2-dG lower down in the active site, in which syn-adenine can pair with adduct-dG via a non-canonical dNTP shape.

DNA‐Dependent DNA Polymerases

This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion-bypass DNA polymerases that play a role in DNA damage repair.

A Dynamic Polymerase Exchange with Escherichia coli DNA Polymerase IV Replacing DNA Polymerase III on the Sliding Clamp*

An assay that measures synchronized, processive DNA replication by Escherichia coli DNA polymerase III holoenzyme was used to reveal replacement of pol III by the specialized lesion bypass DNA polymerase IV when the replicative polymerase is stalled. When idled replication is restarted, a rapid burst of pol III-catalyzed synthesis accompanied by approximately 7-kb full-length products is strongly inhibited by the presence of pol IV. The production of slower-forming, shorter length DNA reflects a rapid takeover of DNA synthesis by pol IV. Here we demonstrate that pol IV rapidly (<15 s) obstructs the stable interaction between pol III* and the beta clamp (the lifetime of the complex is >5 min), causing the removal of pol III* from template DNA. We propose that the rapid replacement of pol III* on the beta clamp with pol IV is mediated by two processes, an interaction between pol IV and the beta clamp and a separate interaction between pol IV and pol III*. This newly discovered property of pol IV facilitates a dynamic exchange between the two free polymerases at the primer terminus. Our study suggests a model in which the interaction between pol III* and the beta clamp is mediated by pol IV to ensure that DNA replication proceeds with minimal interruption.

RecA

RecA

A Water-Mediated and Substrate-Assisted Catalytic Mechanism forSulfolobus solfataricusDNA Polymerase IV

DNA polymerases are enzymes responsible for the synthesis of DNA from nucleotides. Understanding their molecular fundamentals is a prerequisite for elucidating their aberrant activities in diseases such as cancer. Here we have carried out ab initio quantum mechanical/molecular mechanical (QM/MM) studies on the nucleotidyl-transfer reaction catalyzed by the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus, with template guanine and Watson-Crick paired dCTP as the nascent base pair. The results suggested a novel water-mediated and substrate-assisted (WMSA) mechanism: the initial proton transfer to the alpha-phosphate of the substrate via a bridging crystal water molecule is the rate-limiting step, the nucleotidyl-transfer step is associative with a metastable pentacovalent phosphorane intermediate, and the pyrophosphate leaving is facilitated by a highly coordinated proton relay mechanism through mediation of water which neutralizes the evolving negative charge. The conserved carboxylates, which retain their liganding to the two Mg2+ ions during the reaction process, are found to be essential in stabilizing transition states. This WMSA mechanism takes specific advantage of the unique structural features of this low-fidelity lesion-bypass Y-family polymerase, which has a more spacious and solvent-exposed active site than replicative and repair polymerases.

Environmental Stress and Lesion-Bypass DNA Polymerases

In nature, microbes live under a variety of harsh conditions, such as excess DNA damage, starvation, pH shift, or high temperatures. Microbial cells respond to such stressful conditions mostly by switching global patterns of gene expression to relieve the environmental stress. The SOS response, which is induced by DNA damage, is one such global network of gene expression that plays a crucial role in balancing the genomic stability and flexibility that are necessary to adapt to harsh environments. Here, I review the roles of SOS-inducible and noninducible lesion-bypass DNA polymerases in mutagenesis induced by environmental stress, and discuss how these polymerases are coordinated for the replication of damaged chromosomes. Possible contributions of lesion-bypass DNA polymerase in hyperthermophilic archaea, e.g., Sulfolobus solfataricus, to genome maintenance are also discussed.

The Properties of Steric Gate Mutants Reveal Different Constraints within the Active Sites of Y-family and A-family DNA Polymerases

Y-family (lesion-bypass) DNA polymerases show the same overall structural features seen in other members of the polymerase superfamily, yet their active sites are more open, with fewer contacts to the DNA and nucleotide substrates. This raises the question of whether analogous active-site side chains play equivalent roles in the bypass polymerases and their classical DNA polymerase counterparts. In Klenow fragment, an A-family DNA polymerase, the steric gate side chain (Glu710) not only prevents ribonucleotide incorporation but also plays an important role in discrimination against purine-pyrimidine mispairs. In this work we show that the steric gate (Phe12) of the Y-family polymerase Dbh plays a very minor role in fidelity, despite its analogous role in sugar selection. Using ribonucleotide discrimination to report on the positioning of a mispaired dNTP, we found that the pyrimidine of a Pu-dPyTP nascent mispair occupies a similar position to that of a correctly paired dNTP in the Dbh active site, whereas in Klenow fragment the mispaired dNTP sits higher in the active site pocket. If purine-pyrimidine mispairs adopt the expected wobble geometry, the difference between the two polymerases can be attributed to the binding of the templating base, with the looser binding site of Dbh permitting a variety of template conformations with only minimal adjustment at the incoming dNTP. In Klenow fragment the templating base is more rigidly held, so that changes in base pair geometry would affect the dNTP position, allowing the Glu710 side chain to serve as a sensor of nascent mispairs.

Mirror image stereoisomers of the major benzo[ a]pyrene N 2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli

The potent mutagen/carcinogen benzo[ a]pyrene (B[ a]P) is metabolically activated to (+) -anti-B[ a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, −1 frameshifts, etc.) via its major adduct [+ta]-B[ a]P-N 2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5′-T G sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[ a]P-N 2-dG, along with its mirror image adduct [−ta]-B[ a]P-N 2-dG. Each adduct is built into a 5′-T G sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [−ta]-B[ a]P-N 2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[ a]P-N 2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [−ta]-B[ a]P-N 2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[ a]P-N 2-dG requires DNAPs IV and V, [−ta]-B[ a]P-N 2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.