PCNA monoubiquitylation and DNA polymerase eta ubiquitin-binding domain are required to prevent 8-oxoguanine-induced mutagenesis in Saccharomyces cerevisiae

Abstract

7,8-Dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic DNA lesion. In Saccharomyces cerevisiae, the 8-oxoG DNA N-glycosylase (Ogg1) acts as the primary defense against 8-oxoG. Here, we present evidence for cooperation between Rad18-Rad6-dependent monoubiquitylation of PCNA at K164, the damage-tolerant DNA polymerase eta and the mismatch repair system (MMR) to prevent 8-oxoG-induced mutagenesis. Preventing PCNA modification at lysine 164 (pol30-K164R) results in a dramatic increase in GC to TA mutations due to endogenous 8-oxoG in Ogg1-deficient cells. In contrast, deletion of RAD5 or SIZ1 has little effect implying that the modification of PCNA relevant for preventing 8-oxoG-induced mutagenesis is monoubiquitin as opposed to polyubiquitin or SUMO. We also report that the ubiquitin-binding domain (UBZ) of Pol eta is essential to prevent 8-oxoG-induced mutagenesis but only in conjunction with a functional PCNA-binding domain (PIP). We propose that PCNA is ubiquitylated during the repair synthesis reaction after the MMR-dependent excision of adenine incorporated opposite to 8-oxoG. Monoubiquitylation of PCNA would favor the recruitment of Pol eta thereby allowing error-free incorporation of dCMP opposite to 8-oxoG. This study suggests that Pol eta and the post-replication repair (PRR) machinery can also prevent mutagenesis at DNA lesions that do not stall replication forks.

Figure 1.

Expression of the MuY protein of E. coli in S. cerevisiae. WT strain of S. cerevisiae harboring p415-GAL1 or p415-GAL1-mutY was grown at 30°C in YNBGal supplemented medium until OD₆₀₀ = 1.0. Cell free extracts were prepared and assayed for MutY activity using a 34-mer DNA duplex that contained a single A*.8-oxoG pair. Total protein concentration in extracts was as follows: WT/p415-GAL1 (4.2 mg/ml) and WT/p415-GAL1-mutY (3.8 mg/ml). S: 34-mer Substrate, P: 19-mer Product. A*: the A-containing strand was [³²P]-labeled at the 5′-end.

Figure 2.

Ubiquitylation of PCNA in mutator strains of S. cerevisiae. Asynchronous exponentially growing untreated (no damage) cells were allowed to grow at 30°C until OD600 = 1.0. MMS-treated cells were exposed to 0.02% MMS for 90 min at 30°C. Crude extracts from untreated and MMS-treated cells were prepared under denaturing conditions. ^HisPCNA was isolated and its modifications were detected by Western blotting with anti-PCNA and anti-ubiquitin antibodies. Migration of the ubiquitylated forms of PCNA (PCNA-Ub1, PCNA-Ub2 and PCNA-Ubn) and sumoylated forms of PCNA (PCNA-S) are indicated (Figure 2, right). Migration of molecular weight markers (kDa) is indicated (Figure 2, left). (A) WT, ogg1, msh6, msh6 ogg1 ^His-PCNA strains untreated and MMS (0.02%) treated. (B) WT and apn1 apn2 ^His-PCNA strains untreated and MMS (0.02%) treated.

Figure 3.

The UBZ- and PIP-domains of Pol η are required to prevent 8-oxoG-induced mutagenesis. Strains bearing a rad30 deletion (A) or ogg1 rad30 deletions (B) were complemented by expressing a chromosomal version of Rad30-WT, Rad30-D570A, Rad30-FF627/628AA (PIP*), Rad30-(D570A, PIP*) and Rad30-HH568/572AA placed under the control of its natural promoter. Strains are described in ‘Materials and methods’ section and Table 1. Can^R mutation rates were determined as described (Table 2). Δ ogg1 Can^R mutation rate value: 17.4 × 10⁻⁷ (13.4–24.8).

Figure 4.

Physical interactions between Rad30-WT, Rad30-D570A, Rad30-HH568/572AA and PCNA*, Ub*-PCNA* or PCNA*-Ub* fusions. Interactions were monitored in the two-hybrid system, based on fusions to the GAL4 activation (AD) and GAL4 DNA-binding (BD) domains. Truncated RAD30, comprising amino acids 538–632, either WT or mutant (D570A and HH568/572AA) were used. Mutated PCNA (PCNA*) and linear fusions of mutated ubiquitin and PCNA at the N-terminus (Ub*-PCNA*) or the C-terminus (PCNA*-Ub*) were fused to GAL4-AD and GAL4-BD [‘Materials and Methods’ section (37)]. Interactions were scored by growth on plates lacking histidine (-HLW) and plates lacking histidine and adenine (-AHLW). Plates were scored after 3 days at 30°C. (A) Rad30 (538–632) fused to GAL4-AD. (B) Rad30 (538–632) fused to GAL4-BD.