PCNA monoubiquitylation and DNA polymerase eta ubiquitin-binding domain are required to prevent 8-oxoguanine-induced mutagenesis in Saccharomyces cerevisiae.
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.
van der Kemp
Patricia Auffret
PA
CEA, iRCM, UMR217 CNRS Radiobiologie Moléculaire et Cellulaire, 18 route du Panorama, BP6, 92265-Fontenay aux Roses, France.
de Padula
Marcelo
M
Burguiere-Slezak
Guenaelle
G
Ulrich
Helle D
HD
Boiteux
Serge
S
D001665:Binding Sites; D002172:Canavanine / Q000494:pharmacology; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination; D000099270:Y-Family DNA Polymerases
D002172:Canavanine; D045647:DNA Glycosylases; D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C522758:Pol32 protein, S cerevisiae; C024829:8-hydroxyguanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C505160:Ogg1 protein, S cerevisiae; C061738:mutY adenine glycosylase