Article

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

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.

Download full-text

Full-text

Available from: Scott D Mcculloch, Aug 22, 2015
0 Followers
 · 
119 Views
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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 · 2.55 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "The incorporation of 2'-deoxyribonucleotides opposite 8-OH-Gua by human DNA pol η was analyzed in vitro. Human DNA pol η incorporates dATP and dCTP opposite the lesion with comparable efficiencies, but proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A) enhance the correct 8-OH-Gua bypass [25] [26] [27] [28] [29] [30]. The mammalian replicative DNA pols α and δ are error-prone against 8-OH-Gua [27] [28] [42] [43] [44] [45] [46] [47]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C-->T:A and A:T-->C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases eta and zeta by siRNAs enhanced the G:C-->T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases eta and zeta, suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase eta slightly decreased the A:T-->C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases eta and zeta are involved in the bypass of 8-OH-Gua in human cells.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2010; 686(1-2):90-5. DOI:10.1016/j.mrfmmm.2010.02.001 · 4.44 Impact Factor
Show more