Abstract
3-Methyl adenine (3meA), a minor-groove DNA lesion, presents a strong block to synthesis by replicative DNA polymerases (Pols). To elucidate the means by which replication through this DNA lesion is mediated in eukaryotic cells, here we carry out genetic studies in the yeast Saccharomyces cerevisiae treated with the alkylating agent methyl methanesulfonate. From the studies presented here, we infer that replication through the 3meA lesion in yeast cells can be mediated by the action of three Rad6-Rad18-dependent pathways that include translesion synthesis (TLS) by Pol(eta) or -zeta and an Mms2-Ubc13-Rad5-dependent pathway which presumably operates via template switching. We also express human Pols iota and kappa in yeast cells and show that they too can mediate replication through the 3meA lesion in yeast cells, indicating a high degree of evolutionary conservation of the mechanisms that control TLS in yeast and human cells. We discuss these results in the context of previous observations that have been made for the roles of Pols eta, iota, and kappa in promoting replication through the minor-groove N2-dG adducts.
Highlights
Replicative DNA polymerase (Pols) are highly sensitive to geometric distortions in DNA; they are inhibited by the presence of DNA lesions in the template strand
We found that deletion of the REV3 gene, which encodes the catalytic subunit of Pol, confers a higher level of sensitivity to methyl methanesulfonate (MMS) than does deletion of RAD30, encoding Pol, whereas the mms2⌬ mutation confers an intermediate level of MMS sensitivity between that resulting from the rad30⌬ and rev3⌬ mutations, and the rad5⌬ mutation elicits a somewhat higher level of MMS sensitivity than the rev3⌬ mutation (Fig. 1A)
MMS treatment causes the formation of 3-Methyl adenine (3meA) and 7meG in DNA, 3meA presents a strong block to synthesis by the replicative polymerases; the cytotoxicity of MMS results from 3meA and not from the 7meG lesion
Summary
Replicative DNA polymerase (Pols) are highly sensitive to geometric distortions in DNA; they are inhibited by the presence of DNA lesions in the template strand. A number of Pols that promote replication through DNA lesions exist in eukaryotes, and they are highly specialized for the roles they play in translesion synthesis (TLS) [40]. Both yeast and human Pols are highly adept at promoting error-free replication through UV-induced cyclobutane pyrimidine dimers (CPDs) [15, 20, 50, 52], and inactivation of Pol in humans causes the cancer-prone syndrome of the variant form of xeroderma pigmentosum [14, 30]. Pol differs strikingly from Pols and and almost all other Pols in that it incorporates nucleotides opposite template purines with much higher efficiency and fidelity than opposite template pyrimidines [6, 19, 44, 49]. The sequential actions of Pols and can promote replication through the structurally more complex aldehyde products of lipid peroxidation, such as trans-4-hydroxy-2-nonenal (HNE) [55]
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