Deaminated purine bypass by DNA polymerase η.
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ABSTRACT:
SUBMITTER: Andreeva A
PROVIDER: S-EPMC8009656 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The exocyclic amines of nucleobases can undergo deamination by various DNA damaging agents such as reactive oxygen species, nitric oxide, and water. The deamination of guanine and adenine generates the promutagenic xanthine and hypoxanthine, respectively. The exocyclic amines of bases in DNA are hydrogen bond donors, while the carbonyl moiety generated by the base deamination acts as hydrogen bond acceptors, which can alter base pairing properties of the purines. Xanthine is known to base pair with both cytosine and thymine, while hypoxanthine predominantly pairs with cytosine to promote A to G mutations. Despite the known promutagenicity of the major deaminated purines, structures of DNA polymerase bypassing these lesions have not been reported. To gain insights into the deaminated-induced mutagenesis, we solved crystal structures of human DNA polymerase η (polη) catalyzing across xanthine and hypoxanthine. In the catalytic site of polη, the deaminated guanine (i.e., xanthine) forms three Watson-Crick-like hydrogen bonds with an incoming dCTP, indicating the O2-enol tautomer of xanthine involves in the base pairing. The formation of the enol tautomer appears to be promoted by the minor groove contact by Gln38 of polη. When hypoxanthine is at the templating position, the deaminated adenine uses its O6-keto tautomer to form two Watson-Crick hydrogen bonds with an incoming dCTP, providing the structural basis for the high promutagenicity of hypoxanthine.
| S-EPMC8138886 | biostudies-literature
Project description:Oxaliplatin, together with cisplatin, is among the most important drugs used in cancer chemotherapy. Oxaliplatin, which contains a bulky diaminocyclohexane (DACH) moiety, kills cancer cells mainly by producing (DACH)Pt-GpG intrastrand cross-links that impede transcription. The Pt-GpG tolerance by translesion DNA synthesis (TLS) polymerases contributes to the resistance of tumors to platinum-based chemotherapy. In particular, human DNA polymerase η (Polη) readily bypasses Pt-GpG adducts. While many structural studies have addressed how TLS polymerases interact with cisplatin-DNA adducts, a structure of DNA polymerase in complex with oxaliplatin-DNA adducts has not been reported, limiting our understanding of bypass of the bulky (DACH)Pt-GpG lesion by TLS polymerases. Herein, we report the first structure of DNA polymerase bound to oxaliplatinated DNA. We determined a crystal structure of Polη incorporating dCTP opposite the 3'G of the (DACH)Pt-GpG, which provides insights into accurate, efficient bypass of the oxaliplatin-GpG adducts by TLS polymerases. In the catalytic site of Polη, the 3'G of the (DACH)Pt-GpG formed three Watson-Crick hydrogen bonds with incoming dCTP and the primer terminus 3'-OH was optimally positioned for nucleotidyl transfer. To accommodate the bulky (DACH)Pt-GpG lesion, the Val59-Trp64 loop in the finger domain of Polη shifted from the positions observed in the corresponding Polη-cisplatin-GpG and undamaged structures, suggesting that the flexibility of the Val59-Trp64 loop allows the enzyme's bypass of the (DACH)Pt-GpG adducts. Overall, the Polη-oxaliplatin-GpG structure provides a structural basis for TLS-mediated bypass of the major oxaliplatin-DNA adducts and insights into resistance to platinum-based chemotherapy in humans.
| S-EPMC6657808 | biostudies-literature
Project description:Heterocyclic aromatic amines produce bulky C8 guanine lesions in vivo, which interfere and disrupt DNA and RNA synthesis. These lesions are consequently strong replication blocks. In addition bulky adducts give rise to point and frameshift mutations. The translesion synthesis (TLS) DNA polymerase ? is able to bypass slowly C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG and its acetylated analogue mainly in an error-free manner. Replicative polymerases are in contrast fully blocked by the acetylated lesion. Here, we show that TLS efficiency of Pol ? depends critically on the size of the bulky adduct forming the lesion. Based on the crystal structure, we show why the bypass reaction is so difficult and we provide a model for the bypass reaction. In our model, TLS is accomplished without rotation of the lesion into the anti conformation as previously thought.
| S-EPMC2996444 | biostudies-literature
Project description:DNA polymerase eta (pol eta) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol eta also has other cellular roles. Here, we present evidence that pol eta competes with DNA polymerases alpha and delta for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol eta which contains a PCNA-Interacting Protein motif is required for pol eta to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol eta and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.
2018-12-14 | GSE110241 | GEO
Project description:The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.
| S-EPMC2899710 | biostudies-literature
Project description:Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase eta (pol eta) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol eta copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol eta. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol eta and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol eta. We then perform a comprehensive analysis of yeast pol eta fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se.
| S-EPMC2288658 | biostudies-literature
Project description:DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt multiple configurations.
| S-EPMC2570044 | biostudies-literature
Project description:With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B-family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B-family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5'-overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B-family DNA polymerases.
| S-EPMC8596578 | biostudies-literature
Project description:Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.
| S-EPMC7307195 | biostudies-literature
Project description:Etheno (ε)-adducts, e.g., 1,N2-ε-guanine (1,N2-ε-G) and 1,N6-ε-adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N2-ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N2-ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with nonhydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5'-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N2-ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N2-ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication.
| S-EPMC8121704 | biostudies-literature