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Aberrant Repair Initiated by the adenine-DNA Glycosylase Does Not Play a Role in UV-induced Mutagenesis in Escherichia coli

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Aberrant Repair Initiated by the adenine-DNA Glycosylase Does Not Play a Role in UV-induced Mutagenesis in Escherichia coli

Caroline Zutterling et al. PeerJ.

Abstract

Background: DNA repair is essential to counteract damage to DNA induced by endo- and exogenous factors, to maintain genome stability. However, challenges to the faithful discrimination between damaged and non-damaged DNA strands do exist, such as mismatched pairs between two regular bases resulting from spontaneous deamination of 5-methylcytosine or DNA polymerase errors during replication. To counteract these mutagenic threats to genome stability, cells evolved the mismatch-specific DNA glycosylases that can recognize and remove regular DNA bases in the mismatched DNA duplexes. The Escherichia coli adenine-DNA glycosylase (MutY/MicA) protects cells against oxidative stress-induced mutagenesis by removing adenine which is mispaired with 7,8-dihydro-8-oxoguanine (8oxoG) in the base excision repair pathway. However, MutY does not discriminate between template and newly synthesized DNA strands. Therefore the ability to remove A from 8oxoG•A mispair, which is generated via misincorporation of an 8-oxo-2'-deoxyguanosine-5'-triphosphate precursor during DNA replication and in which A is the template base, can induce A•T→C•G transversions. Furthermore, it has been demonstrated that human MUTYH, homologous to the bacterial MutY, might be involved in the aberrant processing of ultraviolet (UV) induced DNA damage.

Methods: Here, we investigated the role of MutY in UV-induced mutagenesis in E. coli. MutY was probed on DNA duplexes containing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproduct (6-4PP). UV irradiation of E. coli induces Save Our Souls (SOS) response characterized by increased production of DNA repair enzymes and mutagenesis. To study the role of MutY in vivo, the mutation frequencies to rifampicin-resistant (RifR) after UV irradiation of wild type and mutant E. coli strains were measured.

Results: We demonstrated that MutY does not excise Adenine when it is paired with CPD and 6-4PP adducts in duplex DNA. At the same time, MutY excises Adenine in A•G and A•8oxoG mispairs. Interestingly, E. coli mutY strains, which have elevated spontaneous mutation rate, exhibited low mutational induction after UV exposure as compared to MutY-proficient strains. However, sequence analysis of RifR mutants revealed that the frequencies of C→T transitions dramatically increased after UV irradiation in both MutY-proficient and -deficient E. coli strains.

Discussion: These findings indicate that the bacterial MutY is not involved in the aberrant DNA repair of UV-induced DNA damage.

Keywords: Aberrant DNA repair; Adenine-DNA glycosylase; Base excision repair; Cyclobutane pyrimidine dimer; Escherichia coli; Nucleotide excision repair; Pyrimidine (6–4) pyrimidone photoproduct; UV-induced mutagenesis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Analysis of the cleavage products generated by T4-PDG and UVDE enzymes.
T4-PDG and UVDE enzymes were acted upon 5′-[32P]-labelled 24 and 30 mer duplex oligonucleotides containing CPD and 6-4PP adducts. Lanes 1, 7 and 10, control non-treated oligonucleotides; lanes 2–3 and 8–9, 24 mer AA•T=T and AA•T-T duplexes incubated either with T4-PDG or UVDE; lanes 11 and 12, 30 mer AA•T=T duplex incubated with T4-PDG and UVDE, respectively; lanes 4–6, 24 mer regular AA•TT duplex incubated with MtbXth, a 3′-5′ exonuclease, to generate size markers. For details see materials and Methods.
Figure 2
Figure 2. Analysis of the cleavage products generated by MutY and UNG when acting upon 24 mer oligonucleotides containing base modifications.
The 5′-[32P]-labelled 24 mer duplex oligonucleotides containing CPD, 6–4PP, mismatches A•G and A•8oxoG and single-stranded 24 mer oligonucleotide containing uracil were incubated with MutY and UNG, respectively. Lanes 1–10, 24 mer duplexes incubated or not with MutY; lanes 11–14, 24 mer single-stranded oligonucleotides containing single Uracil residue, incubated or not with Ung to generate size markers. For details see Materials and Methods.
Figure 3
Figure 3. Analysis of the cleavage products generated by MutY when acting upon 30 and 24 mer duplex oligonucleotides containing either A•G mismatch or CPD adduct.
MutY was acted upon 5′-[32P]-labelled 30 and 24 mer duplex oligonucleotides containing either A•G mismatch or CPD adduct. Lanes 1, 3 and 5, control non-treated 30 mer oligonucleotides; lanes 2, 4 and 6, 30 mer duplexes incubated with MutY; Lanes 7, 9 and 11, control non-treated 24 mer duplexes; lanes 8, 10 and 12, 24 mer duplexes incubated with MutY. For details see materials and Methods.
Figure 4
Figure 4. Graphic representation of the UV-induced increase in mutation frequencies in E. coli cells.
NER-proficient strains were exposed to dose 100–180 J·m−2 UV and NER deficient strains to doses 10 J·m−2 UV, only. Data from at least three experiments were used for statistical analysis.
Figure 5
Figure 5. Graphic representation of the UV-induced increase in mutation frequencies in E. coli strains containing the WT and D138N mutant MutY protein.
NER-proficient AB1157 strain was exposed to 100 J·m−2 UV and NER deficient strains to only 10 J·m−2 UV. Data from at least three experiments were used for statistical analysis.
Figure 6
Figure 6. Distance between C1′ atoms in the adjacent nucleotides.
(A) Graphical representation of the interatomic distances. In 1TTD, 1COC and 355D structures, all distances (except within a CPD in 1TTD) were measured as representative of B-DNA. MutY 5′ and MutY 3′, the distances from C1′ of oxoG to C1′ of 5′- and 3′-adjacent nucleotides, respectively (structures 1RRQ, 1RRS, 1VRL, 3FSP, 5DPK, 3G0Q, 4YOQ, 4YPH and 4YPR). CPD, the distance between two C1′ atoms within a CPD (structures 1N4E, 1SKS, 1SL1, 1SL2, 1TTD, 1VAS, 3MFI, 3MR3, 3MR5, 3MR6, 3PZP, 3SI8, 4A0A, 4A0B, 4A08, 4A09 and 5B24). (B) Close view of a CPD in two representative CPD-containing structures (1TTD: free CPD-containing DNA, 1VAS: CPD-containing DNA from a complex with phage T4 endonuclease V) and a structure of the non-target strand from a complex with G. stearothermophilus MutY (1RRQ). In the 1RRQ structure, the wedging Tyr88 residue is shown. C1′-C1′ distances are indicated.

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References

    1. Abeldenov S, Talhaoui I, Zharkov DO, Ishchenko AA, Ramanculov E, Saparbaev M, Khassenov B. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis. DNA Repair. 2015;33:1–16. doi: 10.1016/j.dnarep.2015.05.007. - DOI - PubMed
    1. Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR, Cheadle JP. Inherited variants of MYH associated with somatic G:C→T:A mutations in colorectal tumors. Nature Genetics. 2002;30(2):227–232. doi: 10.1038/ng828. - DOI - PubMed
    1. Barnes DE, Lindahl T. Repair and genetic consequences of endogenous DNA base damage in mammalian cells. Annual Review of Genetics. 2004;38(1):445–476. doi: 10.1146/annurev.genet.38.072902.092448. - DOI - PubMed
    1. Biertumpfel C, Zhao Y, Kondo Y, Ramon-Maiques S, Gregory M, Lee JY, Masutani C, Lehmann AR, Hanaoka F, Yang W. Structure and mechanism of human DNA polymerase eta. Nature. 2010;465(7301):1044–1048. doi: 10.1038/nature09196. - DOI - PMC - PubMed
    1. Bulychev NV, Varaprasad CV, Dorman G, Miller JH, Eisenberg M, Grollman AP, Johnson F. Substrate specificity of Escherichia coliMutY protein. Biochemistry. 1996;35(40):13147–13156. - PubMed

Grant support

This work was supported by grants to Murat Saparbaev from la Ligue National Contre le Cancer “Equipe Labellisee,” Electricité de France (RB 2017) and French National Center for Scientific Research (PRC CNRS/RFBR n1074 REDOBER); and to Bakhyt T. Matkarimov from NU ORAU (https://nu.edu.kz/) and Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan, Program 0115RK03029; and to Dmitry O. Zharkov from the Russian Ministry of Science and Education (6.5773.2017/6.7) and Russian Science Foundation (17-14-01190), and to Amangeldy K. Bissenbaev from the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan [grant No. AP05131598], and to Nicolas E. Geacintov from US NIEHS Grant ES024050. Didier Gasparutto received support from the Arcane Labex program, funded by the French National Research Agency (ARCANE project no. ANR-12-LABX-003). Ibtissam Talhaoui was supported by postdoctoral fellowships from the Fondation ARC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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