A role for yeast and human translesion synthesis DNA polymerases in promoting replication through 3-methyl adenine

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.

FIG. 1.

MMS sensitivity of yeast strains carrying deletions of genes belonging to the RAD6 epistasis group. Cells were treated with the indicated concentrations of MMS (percent [vol/vol]) at 30°C for 20 min, followed by inactivation of MMS with sodium thiosulfate. Appropriate dilutions of cells were plated on YPD for viability determinations. The survival curves represent an average of at least three experiments for each strain. WT, wild type. (A) MMS sensitivity of strains deleted for the TLS polymerases η and ζ and for the MMS2 and RAD5 genes, involved in postreplicational repair. (B) MMS sensitivity of the mag1Δ strain in combination with deletions of genes encoding the TLS polymerase η or ζ or genes that function in PRR. (C) MMS sensitivity of the mag1Δ strain in combination with deletions of genes, so that all three pathways of Rad6-Rad18-dependent lesion bypass have been inactivated.

FIG. 2.

MMS-induced can1^r mutations in various mutant strains. Cells were treated for 20 min at 30°C with the indicated MMS concentrations. Following inactivation of MMS, cells were spread onto the surface of YPD plates for viability determinations and onto synthetic complete medium plates containing canavanine but lacking arginine for determining the frequency of can1^r mutations. Each histogram represents the average of two to three experiments. WT, wild type.

FIG. 3.

Enhanced MMS resistance conferred upon mag1Δ and mag1Δ rad30Δ strains by human Pols ι and κ. For each strain, a sample of cells containing a given 10-fold serial dilution was pipetted into a well of a 96-well microtiter dish as described in Materials and Methods. Cells were transferred to YPD plates containing different MMS concentrations and photographed after 2 days of incubation at 30°C.

FIG. 4.

Modeling of 3meA in the active sites of Polκ, Polι, and Polη. 3meA was modeled in place of A at the templating base (insertion) or in place of G at the postinsertion template residue (extension) for each polymerase. Surface representations of Polκ (upper panels; PDB accession no. 2OH2), Polι (middle panels; PDB accession no. 2FLL), and Polη (lower panels; PDB accession no. 1JIH) are shown in cyan, magenta, and yellow, respectively. The methyl group of 3meA is shown in green. The DNA from the Polκ structure was modeled into Polη based on the alignment of the Polκ and Polη catalytic residues. The incoming dTTP is shown opposite the 3meA at position T₀. For modeling 3meA at the T₋₁ position, the G residue present in the structures was replaced by 3meA; the original cognate C residue is shown to provide a reference point for the base pair; the next template residue and incoming dTTP are shown in gray. The thumb region of each polymerase was removed to provide a clear view of the active sites.