Nucleic Acids Research Advance Access published online on February 14, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn058
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Molecular Biology |
Eukaryotic Y-family polymerases bypass a 3-methyl-2'-deoxyadenosine analog in vitro and methyl methanesulfonate-induced DNA damage in vivo
1Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371 and 2Berry & Associates, Inc., 2434 Bishop Circle East, Dexter, MI 48130, USA
*To whom correspondence should be addressed. Tel: +1 301 217 4040; Fax: +1 301 217 5815; Email: woodgate{at}nih.gov
Received November 9, 2007. Revised January 26, 2008. Accepted January 29, 2008.
N3-methyl-adenine (3MeA) is the major cytotoxic lesion formed in DNA by SN2 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 
and 
. In contrast, human polymerases 
, 
and 
, as well as Saccharomyces cerevisiae pol 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, pol (Rad30) contributes to the survival of cells exposed to methyl methanesulfonate (MMS) and in the absence of Mag1, Rad30 and Rev3, human polymerases , and are capable of restoring MMS-resistance to the normally MMS-sensitive strain.