The Role of DNA Mismatch Repair and Recombination in the Processing of DNA Alkylating Damage in Living Yeast Cells

Hernan Flores-Rozas^1,2*, Lahcen Jaafar², Ling Xia²

¹College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, USA.
²Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, USA.

DOI: 10.4236/abb.2015.66040                

Abstract

It is proposed that mismatch repair (MMR) mediates the cytotoxic effects of DNA damaging agents by exerting a futile repair pathway which leads to double strand breaks (DSBs). Previous reports indicate that the sensitivity of cells defective in homologous recombination (HR) to DNA alkylation is reduced by defects in MMR genes. We have assessed the contribution of different MMR genes to the processing of alkylation damage in vivo. We have directly visualized recombination complexes formed upon DNA damage using fluorescent protein (FP) fusions. We find that msh6 mutants are more resistant than wild type cells to MNNG, and that an msh6 mutation rescues the sensitivity of rad52 strains more efficiently than an msh3 mutation. Analysis of RAD52-GFP tagged strains indicate that MNNG increases repair foci formation, and that the inactivation of the MHS2 and MSH6 genes but not the MSH3 gene result in a reduction of the number of foci formed. In addition, in the absence of HR, NHEJ could process the MNNG-induced DSBs as indicated by the formation of NHEJ-GFP tagged foci. These data suggest that processing of the alkylation damage by MMR, mainly by MSH2-MSH6, is required for recruitment of recombination proteins to the damage site for repair.

Keywords

DNA Mismatch Repair, Recombination, DNA Damage, Non-Homologous End Joining

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Chen, W. and Jinks-Robertson, S. (1998) Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast. Molecular and Cellular Biology, 18, 6525-6537.
[2]	Kolodner, R.D. and Marsischky, G.T. (1999) Eukaryotic DNA Mismatch Repair. Current Opinion in Genetics & Development, 9, 89-96. http://dx.doi.org/10.1016/S0959-437X(99)80013-6
[3]	Jiricny, J. (2006) The Multifaceted Mismatch-Repair System. Nature Reviews Molecular Cell Biology, 7, 335-346. http://dx.doi.org/10.1038/nrm1907
[4]	Eshleman, J.R., Lang, E.Z., Bowerfind, G.K., Parsons, R., Vogelstein, B., Willson, J.K., Veigl, M.L., Sedwick, W.D. and Markowitz, S.D. (1995) Increased Mutation Rate at the Hprt Locus Accompanies Microsatellite Instability in Colon Cancer. Oncogene, 10, 33-37.
[5]	Iyer, R.R., Pluciennik, A., Burdett, V. and Modrich, P.L. (2006) DNA Mismatch Repair: Functions and Mechanisms. Chemical Reviews, 106, 302-323. http://dx.doi.org/10.1021/cr0404794
[6]	Pegg, A.E. (2000) Repair of O6-Alkylguanine by Alkyltransferases. Mutation Research/Reviews in Mutation Research, 462, 83-100. http://dx.doi.org/10.1016/S1383-5742(00)00017-X
[7]	Xiao, W., Derfler, B., Chen, J. and Samson, L. (1991) Primary Sequence and Biological Functions of a Saccharomyces cerevisiae O6-Methylguanine/O4-Methylthymine DNA Repair Methyltransferase Gene. EMBO Journal, 10, 2179-2186.
[8]	Swann, P.F. (1990) Why Do O6-Alkylguanine and O4-Alkylthymine Miscode? The Relationship between the Structure of DNA Containing O6-Alkylguanine and O4-Alkylthymine and the Mutagenic Properties of These Bases. Mutation Research, 233, 81-94. http://dx.doi.org/10.1016/0027-5107(90)90153-U
[9]	Cejka, P. and Jiricny, J. (2008) Interplay of DNA Repair Pathways Controls Methylation Damage Toxicity in Saccharomyces cerevisiae. Genetics, 179, 1835-1844. http://dx.doi.org/10.1534/genetics.108.089979
[10]	Cejka, P., Stojic, L., Mojas, N., Russell, A.M., Heinimann, K., Cannavo, E., di Pietro, M., Marra, G. and Jiricny, J. (2003) Methylation-Induced G(2)/M Arrest Requires a Full Complement of the Mismatch Repair Protein hMLH1. Embo Journal, 22, 2245-2254. http://dx.doi.org/10.1093/emboj/cdg216
[11]	Pegg, A.E., Dolan, M.E. and Moschel, R.C. (1995) Structure, Function, and Inhibition of O6-Alkylguanine-DNA Alkyltransferase. Progress in Nucleic Acid Research and Molecular Biology, 51, 167-223. http://dx.doi.org/10.1016/S0079-6603(08)60879-X
[12]	Haracska, L., Prakash, S. and Prakash, L. (2000) Replication past O(6)-Methylguanine by Yeast and Human DNA Polymerase Eta. Molecular and Cellular Biology, 20, 8001-8007. http://dx.doi.org/10.1128/MCB.20.21.8001-8007.2000
[13]	York, S.J. and Modrich, P. (2006) Mismatch Repair-Dependent Iterative Excision at Irreparable O6-Methylguanine Lesions in Human Nuclear Extracts. Journal of Biological Chemistry, 281, 22674-22683. http://dx.doi.org/10.1074/jbc.M603667200
[14]	Karran, P. (2001) Mechanisms of Tolerance to DNA Damaging Therapeutic Drugs. Carcinogenesis, 22, 1931-1937. http://dx.doi.org/10.1093/carcin/22.12.1931
[15]	Karran, P. and Hampson, R. (1996) Genomic Instability and Tolerance to Alkylating Agents. Journal of Cancer Survivorship, 28, 69-85.
[16]	van Gent, D.C., Hoeijmakers, J.H. and Kanaar, R. (2001) Chromosomal Stability and the DNA Double-Stranded Break Connection. Nature Reviews Genetics, 2, 196-206. http://dx.doi.org/10.1038/85243
[17]	Sonoda, E., Hochegger, H., Saberi, A., Taniguchi, Y. and Takeda, S. (2006) Differential Usage of Non-Homologous End-Joining and Homologous Recombination in Double Strand Break Repair. DNA Repair, 5, 1021-1029. http://dx.doi.org/10.1016/j.dnarep.2006.05.022
[18]	Jeggo, P.A. (1998) Identification of Genes Involved in Repair of DNA Double-Strand Breaks in Mammalian Cells. Radiation Research, 150, S80-S91. http://dx.doi.org/10.2307/3579810
[19]	Sung, P., Trujillo, K.M. and Van Komen, S. (2000) Recombination Factors of Saccharomyces cerevisiae. Mutation Research, 451, 257-275. http://dx.doi.org/10.1016/S0027-5107(00)00054-3
[20]	van den Bosch, M., Lohman, P.H. and Pastink, A. (2002) DNA Double-Strand Break Repair by Homologous Recombination. Journal of Biological Chemistry, 383, 873-892.
[21]	Haber, J.E. (1995) In Vivo Biochemistry: Physical Monitoring of Recombination Induced by Site-Specific Endonucleases. Bioessays, 17, 609-620. http://dx.doi.org/10.1002/bies.950170707
[22]	Symington, L.S. (2002) Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair. Microbiology and Molecular Biology Reviews, 66, 630-670. http://dx.doi.org/10.1128/MMBR.66.4.630-670.2002
[23]	Daley, J.M., Palmbos, P.L., Wu, D. and Wilson, T.E. (2005) Nonhomologous End Joining in Yeast. Annual Review of Genetics, 39, 431-451. http://dx.doi.org/10.1146/annurev.genet.39.073003.113340
[24]	Dudasova, Z., Dudas, A. and Chovanec, M. (2004) Non-Homologous End-Joining Factors of Saccharomyces cerevisiae. FEMS Microbiology Reviews, 28, 581-601. http://dx.doi.org/10.1016/j.femsre.2004.06.001
[25]	Martin, S.G., Laroche, T., Suka, N., Grunstein, M. and Gasser, S.M. (1999) Relocalization of Telomeric Ku and SIR Proteins in Response to DNA Strand Breaks in Yeast. Cell, 97, 621-633. http://dx.doi.org/10.1016/S0092-8674(00)80773-4
[26]	Chen, L., Trujillo, K., Ramos, W., Sung, P. and Tomkinson, A.E. (2001) Promotion of Dnl4-Catalyzed DNA End-Joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 Complexes. Molecular Cell, 8, 1105-1115. http://dx.doi.org/10.1016/S1097-2765(01)00388-4
[27]	Alani, E.R., Reenan, A. and Kolodner, R.D. (1994) Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces cerevisiae. Genetics, 137, 19-39.
[28]	Tishkoff, D.X., Filosi, N., Gaida, G.M. and Kolodner, R.D. (1997) A Novel Mutation Avoidance Mechanism Dependent on Saccharomyces cerevisiae RAD27 Is Distinct from DNA Mismatch Repair. Cell, 88, 253-263. http://dx.doi.org/10.1016/S0092-8674(00)81846-2
[29]	Redon, C., Pilch, D.R. and Bonner, W.M. (2006) Genetic Analysis of Saccharomyces cerevisiae H2A Serine 129 Mutant Suggests a Functional Relationship between H2A and the Sister-Chromatid Cohesion Partners Csm3-Tof1 for the Repair of Topoisomerase I-Induced DNA Damage. Genetics, 172, 67-76. http://dx.doi.org/10.1534/genetics.105.046128
[30]	Lisby, M., Rothstein, R. and Mortensen, U.H. (2001) Rad52 Forms DNA Repair and Recombination Centers during S Phase. Proceedings of the National Academy of Sciences of the United States of America, 98, 8276-8282. http://dx.doi.org/10.1073/pnas.121006298
[31]	Takahashi, A. and Ohnishi, T. (2005) Does γH2AX Foci Formation Depend on the Presence of DNA Double Strand Breaks? Cancer Letters, 229, 171-179. http://dx.doi.org/10.1016/j.canlet.2005.07.016
[32]	Nakamura, T.M., Du, L.-L., Redon, C. and Russell, P. (2004) Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast. Molecular and Cellular Biology, 24, 6215-6230. http://dx.doi.org/10.1128/MCB.24.14.6215-6230.2004