Human Y-family DNA Polymerases Research Articles

Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells.

Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of 10 human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1-445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants (kcat/Km) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I, and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H2O2 in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H2O2 sensitivity in POLI-deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H2O2 sensitivity in POLI-deficient cells, while the R209Q, K228I, and Q386R variants, all impaired in polymerase and dRP lyase activity, failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.

Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol κ are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol κ whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol κ variants generally fall into three categories: similar activity to wild-type (WT) pol κ (L21F, I39T, P169T, F192C, and E292K), more active than WT pol κ (S423R), and less active than pol κ (R219I, R298H, and Y432S). Of these, only pol κ variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol κ with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol κ with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol κ adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.

Structural basis for proficient nucleotide incorporation across a major cisplatin‐DNA lesion by human DNA polymerase kappa (polκ)

Cisplatin (cis‐diamminedichloroplatinum) is a widely used chemotherapeutic compound. It reacts with the N7 atoms of adjacent guanines in DNA to form the Pt‐1,2‐d(GpG) intrastrand cross‐link (Pt‐GG) as a major product. This bulky cisplatin adduct blocks DNA replication by replicative polymerases, thereby promoting apoptosis of rapidly dividing cancer cells. However, cancer cells have developed resistance to cisplatin, partly because of the bypass of cisplatin adducts by specialized DNA polymerases (mostly belonging to the Y‐family) through translesion synthesis (TLS). The human Y‐family DNA polymerase eta (polη) is very efficient in replicating opposite the Pt‐GG lesions, but not in extending after this adduct. Recently, it was shown that human Y‐family DNA polymerase kappa (polκ) is indispensable for DNA repair synthesis in dorsal root ganglion neurons exposed to cisplatin. Polκ gene knockdown experiments in human cells have also indicated the involvement of polκ in TLS of the Pt‐GG lesion. In this study, we investigated the bypass potential and nucleotide incorporation preference of polκ opposite DNA containing the Pt‐GG adduct and determined two crystal structures of polκ complexed with such DNA. The ternary complex structures represent two consecutive stages of lesion bypass: nucleotide insertion opposite the 5′G (Pt‐GG2) and primer extension immediately after the lesion (Pt‐GG3). Our in vitro DNA replication assays using polκ and DNA containing the Pt‐GG lesion indicate that polκ inserts the correct dCTP opposite the 5′G of Pt‐GG (Pt‐GG2), when a nucleotide is already inserted opposite the 3′G (Pt‐GG1). Polκ also inserts the correct dATP opposite the T base immediately after the Pt‐GG (Pt‐GG3). However, polκ is not able to insert a nucleotide opposite the 3′G of Pt‐GG (Pt‐GG1). On the other hand, polη is very efficient in replicating the Pt‐GG lesion at two insertion stages (Pt‐GG1 and Pt‐GG2), but the enzyme is blocked at the extension stage (Pt‐GG3). In terms of fidelity, we found that polκ has the fewest mis‐insertions compared to two other human Y‐family polymerases polη and polι. Our steady‐state kinetic analysis showed that polκ incorporates dCTP and dATP opposite the 5′G of Pt‐GG (Pt‐GG2) and T template 5′ to the lesion (Pt‐GG3), respectively, with very similar relative efficiencies of 0.15–0.20, which are only ~5 fold lower than for normal DNA replication (GG2 and GG3). Our biochemical data indicate that polκ is very proficient and accurate in inserting a nucleotide after the first G of the Pt‐GG lesion. The structures revealed that the accuracy is achieved by stably accommodating the bases containing the cisplatin adducts in the enzyme's active site for proper Watson‐Crick base pairing with the incoming nucleotide. Together, our biochemical and structural data provide the molecular basis for the accurate and proficient incorporation by polκ of nucleotides opposite the cisplatin adduct and suggest an important role of polκ as a second polymerase (extender) in the efficient bypass of the Pt‐GG lesion in vivo. This work holds promise for polκ as a potential target for drug design, along with polη, which together could improve the efficacy of cisplatin treatment for cancer therapy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N6-furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N2-furfuryl-dG or N6-furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N2-furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

Structural basis of accurate replication beyond a bulky major benzo[a]pyrene adduct by human DNA polymerase kappa

Human Y-family DNA polymerase kappa (polκ) is specialized to bypass bulky lesions in DNA in an error-free way, thus protecting cells from carcinogenic bulky DNA adducts. Benzo[a]pyrene (BP) is one of the most ubiquitous polycyclic aromatic hydrocarbons and an environmental carcinogen. BP covalently modifies DNA and generates mutagenic, bulky adducts. The major BP adduct formed in cells is 10S (+)-trans-anti-BP-N2-dG adduct (BP-dG), which is associated with cancer. The molecular mechanism of how polκ replicates BP-dG accurately is not clear. Here we report the structure of polκ captured at the lesion-extension stage: the enzyme is extending the primer strand after the base pair containing the BP-dG adduct in the template strand at the −1 position. Polκ accommodates the BP adduct in the nascent DNA’s minor groove and keeps the adducted DNA helix in a B-form. Two water molecules cover the edge of the minor groove of the replicating base pair (0 position), which is secured by the BP ring in the −1 position in a 5′ orientation. The 5′ oriented BP adduct keeps correct Watson-Crick base pairing in the active site and promotes high fidelity replication. Our structural and biochemical data reveal a unique molecular basis for accurate DNA replication right after the bulky lesion BP-dG.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.

Mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass catalyzed by human Y-family DNA polymerases.

One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPolη), iota (hPolι), kappa (hPolκ), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPolη, hRev1, hPolκ, and hPolι to bypass 50% of the 8-oxodG lesions encountered (t50bypass) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPolη bypassed 8-oxodG most efficiently, hPolη correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPolι bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPolκ was most accurate opposite 8-oxodG (92.3%), whereas hPolκ alone was the least accurate (18.5%). The t50bypass value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPolη alone, suggesting that hPolη outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.

Identification and characterization of novel small molecule inhibitors of the human Y‐family DNA polymerases

Specialized Y‐family DNA polymerases are responsible for bypass of replication blocks, such as DNA adducts or G‐quadruplexes. Misregulation of Y‐family members is associated with many tumor types. They are highly successful in performing their role due to certain unique structural and biochemical properties. These same properties, however also make these enzymes more error‐prone and capable of producing mutations. This phenomenon can be coopted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments. Thus, these enzymes represent potential targets for adjuvant cancer therapies. Recently, the inhibitor of 5‐lipoxygenase activating protein, MK886, was discovered as one of the more potent inhibitors of a specialized DNA polymerase hpol κ. In our study, we investigated the specificity and mechanism of inhibition of MK886 against other human Y‐family DNA polymerases, as well as a model B‐family DNA polymerase. We have expanded our studies to include inhibitors that were identified by screening a targeted library of compounds synthesized by the laboratory of Prof. Peter Crooks (UAMS). Kinetic analyses and in silico docking studies were used to determine the mechanism of polymerase inhibition by these novel compounds. Our results provide a framework for developing potent inhibitors of translesion DNA polymerases.Supported in part by USPHS R00 GM084460 (R.L.E.)

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.

Replication of a carcinogenic nitropyrene DNA lesion by human Y-family DNA polymerase

Nitrated polycyclic aromatic hydrocarbons are common environmental pollutants, of which many are mutagenic and carcinogenic. 1-Nitropyrene is the most abundant nitrated polycyclic aromatic hydrocarbon, which causes DNA damage and is carcinogenic in experimental animals. Error-prone translesion synthesis of 1-nitropyrene–derived DNA lesions generates mutations that likely play a role in the etiology of cancer. Here, we report two crystal structures of the human Y-family DNA polymerase iota complexed with the major 1-nitropyrene DNA lesion at the insertion stage, incorporating either dCTP or dATP nucleotide opposite the lesion. Polι maintains the adduct in its active site in two distinct conformations. dCTP forms a Watson–Crick base pair with the adducted guanine and excludes the pyrene ring from the helical DNA, which inhibits replication beyond the lesion. By contrast, the mismatched dATP stacks above the pyrene ring that is intercalated in the helix and achieves a productive conformation for misincorporation. The intra-helical bulky pyrene mimics a base pair in the active site and facilitates adenine misincorporation. By structure-based mutagenesis, we show that the restrictive active site of human polη prevents the intra-helical conformation and A-base misinsertions. This work provides one of the molecular mechanisms for G to T transversions, a signature mutation in human lung cancer.

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N(2),3-Ethenoguanine (N(2),3-εG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2'-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2'-fluoro-N(2),3-ε-2'-deoxyarabinoguanosine to investigate the miscoding potential of N(2),3-εG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N(2),3-εG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N(2),3-εG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N(2),3-εG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N(2),3-εG:dCTP base pair, whereas only one appears to be present in the case of the N(2),3-εG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N(2),3-εG.

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans.

Kinetic Analysis of the Bypass of a Bulky DNA Lesion Catalyzed by Human Y-Family DNA Polymerases

1-Nitropyrene (1-NP), a mutagen and potential carcinogen, is the most abundant nitro polyaromatic hydrocarbon in diesel exhaust, which reacts with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)). If not repaired, this DNA lesion is presumably bypassed in vivo by any of human Y-family DNA polymerases kappa (hPolκ), iota (hPolι), eta (hPolη), and Rev1 (hRev1). Our running start assays demonstrated that each of these enzymes was indeed capable of traversing a site-specifically placed dG(AP) on a synthetic DNA template but that hRev1 was stopped after lesion bypass. The time required to bypass 50% of the dG(AP) sites (t(50)(bypass)) encountered by hPolη, hPolκ, and hPolι was determined to be 2.5 s, 4.1 s, and 106.5 s, respectively. The efficiency order of catalyzing translesion synthesis of dG(AP) (hPolη > hPolκ > hPolι ≫ hRev1) is the same as the order for these human Y-family enzymes to elongate undamaged DNA. Although hPolη bypassed dG(AP) efficiently, replication by both hPolκ and hPolι was strongly stalled at the lesion site and at a site immediately downstream from dG(AP). By employing presteady state kinetic methods, a kinetic basis was established for polymerase pausing at these DNA template sites. Besides efficiency of bypass, the fidelity of those low-fidelity polymerases at these pause sites was also significantly decreased. Thus, if the translesion DNA synthesis of dG(AP)in vivo is catalyzed by a human Y-family DNA polymerase, e.g., hPolη, the process is certainly mutagenic.

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι’s biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O 8 atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t50bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t50bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.

In Vitro Bypass of the Major Malondialdehyde- and Base Propenal-Derived DNA Adduct by Human Y-family DNA Polymerases κ, ι, and Rev1

3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one (M1dG) is the major adduct derived from the reaction of DNA with the lipid peroxidation product malondialdehyde and the DNA peroxidation product base propenal. M1dG is mutagenic in Escherichia coli and mammalian cells, inducing base-pair substitutions (M1dG → A and M1dG → T) and frameshift mutations. Y-family polymerases may contribute to the mutations induced by M1dG in vivo. Previous reports described the bypass of M1dG by DNA polymerases η and Dpo4. The present experiments were conducted to evaluate bypass of M1dG by the human Y-family DNA polymerases κ, ι, and Rev1. M1dG was incorporated into template-primers containing either dC or dT residues 5′ to the adduct, and the template-primers were subjected to in vitro replication by the individual DNA polymerases. Steady-state kinetic analysis of single nucleotide incorporation indicates that dCMP is most frequently inserted by hPol κ opposite the adduct in both sequence contexts, followed by dTMP and dGMP. dCMP and dTMP were most frequently inserted by hPol ι, and only dCMP was inserted by Rev1. hPol κ extended template-primers in the order M1dG:dC > M1dG:dG > M1dG:dT ∼ M1dG:dA, but neither hPol ι nor Rev1 extended M1dG-containing template-primers. Liquid chromatography−mass spectrometry analysis of the products of hPol κ-catalyzed extension verified this preference in the 3′-GXC-5′ template sequence but revealed the generation of a series of complex products in which dAMP is incorporated opposite M1dG in the 3′-GXT-5′ template sequence. The results indicate that DNA hPol κ or the combined action of hPol ι or Rev1 and hPol κ bypass M1dG residues in DNA and generate products that are consistent with some of the mutations induced by M1dG in mammalian cells.

Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.