Abstract
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.