The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases and

Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences Research, NC 27709, USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2009; 37(9):2830-40. DOI: 10.1093/nar/gkp103
Source: PubMed

ABSTRACT A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols delta and eta. Yeast Pol delta and yeast Pol eta both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol eta is 10-fold more efficient than Pol delta, and following bypass Pol eta switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol eta is at least 10-fold more accurate than yeast Pol delta during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol eta in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol eta. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol eta. The fact that yeast and mammalian Pol eta have intrinsically different catalytic properties has potential biological implications.

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Available from: Scott D Mcculloch, Sep 26, 2015
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    • "Because H2O2 induces PCNA ubiquitination and as Polη can replicate DNA templates containing oxidative DNA lesions, such as 8-oxodG and AP sites (28,30,44,45), we hypothesized that Polη-mediated TLS was the likely mechanism by which Rad18 suppressed ATM activation in H2O2-treated cells. Consistent with a role of TLS in repressing ATM, partial downregulation of PCNA using siRNA also exacerbated ATM S1981 phosphorylation after H2O2 treatment (Figure 4A). "
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    ABSTRACT: The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
    Nucleic Acids Research 01/2013; 41(4). DOI:10.1093/nar/gks1325 · 9.11 Impact Factor
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    • "Commonly encountered base modifications such as 8-oxo- deoxyguanine or 6-methyl-deoxyguanine can be used as templates by a replicating Pol d with reduced efficiency, but the altered base pairing properties of these lesions usually results in a mispair that pol d is not able to extend [Fazlieva et al., 2009; McCulloch et al., 2009]. When Pol d encounters an abasic site, it has a tendency to incorporate an adenine, possibly due to a highly conserved tyrosine (Y708) which acts as a mock template, and which has a more favorable electrostatic interaction with adenine [Schaaper et al., 1983; Obeid et al., 2010]. "
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    ABSTRACT: The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ϵ on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of pol δ in replication fidelity and genome maintenance. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 12/2012; 53(9). DOI:10.1002/em.21745 · 2.63 Impact Factor
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    • "Furthermore, this enzyme was reportedly able to bypass not only UV-induced lesions but also various other lesions induced by both environmental and endogenous reactive oxygen species. For instance, HsPolη bypasses 7,8-dihydro- 8-oxoguanine (8-oxoG), 5R-thymine glycol (5R-Tg), and 5S- thymine glycol (5S-Tg), but not an apurinic/apyrimidinic (AP) site [13] [14] [15] [16] [17] [18] [19], although these lesions, which have small alterations in their chemical structures, are mainly repaired by BER in vivo. Thus, these findings indicate that HsPolη also plays an important function in the replication of DNA containing ROS-induced lesions in vivo. "
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    ABSTRACT: Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents.
    Journal of nucleic acids 09/2010; 2010. DOI:10.4061/2010/701472
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