Eukaryotic Y-family polymerases bypass a 3-methyl-2'-deoxyadenosine analog in vitro and methyl methanesulfonate-induced DNA damage in vivo.
N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by S(N)2 methylating agents. The lesion presumably blocks progression of cellular replicases because the N3-methyl group hinders interactions between the polymerase and the minor groove of DNA. However, this hypothesis has yet to be rigorously proven, as 3MeA is intrinsically unstable and is converted to an abasic site, which itself is a blocking lesion. To circumvent these problems, we have chemically synthesized a 3-deaza analog of 3MeA (3dMeA) as a stable phosphoramidite and have incorporated the analog into synthetic oligonucleotides that have been used in vitro as templates for DNA replication. As expected, the 3dMeA lesion blocked both human DNA polymerases alpha and delta. In contrast, human polymerases eta, iota and kappa, as well as Saccharomyces cerevisiae poleta were able to bypass the lesion, albeit with varying efficiencies and accuracy. To confirm the physiological relevance of our findings, we show that in S. cerevisiae lacking Mag1-dependent 3MeA repair, poleta (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases eta, iota and kappa are capable of restoring MMS-resistance to the normally MMS-sensitive strain.
Plosky
Brian S
BS
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA.
Frank
Ekaterina G
EG
Berry
David A
DA
Vennall
Graham P
GP
McDonald
John P
JP
Woodgate
Roger
R
D000225:Adenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009153:Mutagens / Q000633:toxicity; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D000099270:Y-Family DNA Polymerases
D000225:Adenine; D045647:DNA Glycosylases; D004259:DNA-Directed DNA Polymerase; D008741:Methyl Methanesulfonate; D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; C528907:3-deaza-3-methyladenine; C092602:REV3 protein, S cerevisiae; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C445668:MAG1 protein, S cerevisiae