Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae

Abstract

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities--a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions--an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct--we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.

Figure 1.—

Plasmids used for TLS assays. (A) Plasmid used for TLS opposite an AP site. The plasmid carries an AP site in the leading or the lagging DNA strand. TLS through the AP site results in Ura+ cells and the frequency of Ura+ cells among Trp+ cells reflects the TLS frequency. (B) Plasmid used for TLS opposite a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct. In this plasmid, the DNA lesion is contained within a heteroduplex sequence (see C) which allows for the detection of TLS events as follows. For each strain, individual colonies are probed with ³²P-labeled oligonucleotides that specifically hybridize with either the lesion-containing target strand or the marker strand. Colonies that hybridize with the target strand are scored as TLS events whereas colonies that hybridize with the marker strand could reflect lesion bypass by a damage avoidance (DA) pathway. For the UV photoproducts, the molecular nature of the TLS event (i.e., error free vs. mutagenic) is determined following isolation of the plasmid DNA from TLS-positive yeast colonies, transformation into Escherichia coli, and sequencing. For the G-AAF adduct, the total extent of TLS is determined by colony hybridization as described above, while mutagenic TLS is scored directly in yeast by overlaying the transformation plates with X-Gal-containing agarose (Bresson and Fuchs 2002). Indeed, the frameshift mutations induced by the G-AAF adduct (+1 and −1 in the GTTT and GCCC context, respectively) restore the lacZ gene reading frame and appear as blue yeast colonies. (C) The heteroduplex region containing the DNA lesion and the sequence changes resulting from the error-free and mutagenic TLS events through the lesions carried on the plasmid shown in B.

Figure 2.—

Rad5 directly binds to Rev1 but not to Polζ. Yeast Rev1 was mixed and incubated with GST–Rad5 (lanes 1–4) or with GST protein alone (lanes 5–8), and Rad5 was mixed and incubated with GST–Polζ (Rev3–Rev7) (lanes 9–12). One microgram of each protein was used in this study. After incubation, samples were bound to glutathione-sepharose beads for 1 hr, followed by multiple washings with buffer I containing 150 mm NaCl and elution of the bound proteins with SDS-sample buffer. Aliquots of each sample before addition to the beads (L), the flow through fraction (F), last washing fraction (W), and the eluted proteins (E) were analyzed on an SDS-12% polyacrylamide gel developed with Coomassie blue.