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

Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Health Sciences Research, NC 27709, USA.
Nucleic Acids Research (Impact Factor: 8.81). 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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    ABSTRACT: The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh(-/-)) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh(-/-) epidermis and dermis than in the wild-type (Polh(+/+)) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh(-/-) mice were determined and compared with mutant sequences from irradiated Polh(+)(/)(+) mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C→T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5'-TCG-3' sequence context than at the other dipyrimidine contexts in the Polh(+/+) epidermis, whereas the mutations were induced remarkably and exclusively at the 3'-cytosine of almost all dipyrimidine contexts with no preference for 5'-TCG-3' in the Polh(-/-) epidermis. In addition, in Polh(-/-) mice, a small but remarkable fraction of G→T transversions was also observed exclusively at the 3'-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh(+/+) genotype.
    DNA Repair 08/2014; 22C:112-122. DOI:10.1016/j.dnarep.2014.07.012 · 3.36 Impact Factor
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    ABSTRACT: Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated. Copyright © 2015. Published by Elsevier B.V.
    DNA Repair 02/2015; DOI:10.1016/j.dnarep.2015.02.006 · 3.36 Impact Factor
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    ABSTRACT: DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.
    PLoS ONE 05/2014; 9(5):e97382. DOI:10.1371/journal.pone.0097382 · 3.53 Impact Factor

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