Contributions of ubiquitin- and PCNA-binding domains to the activity of Polymerase eta in Saccharomyces cerevisiae.
Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase eta (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase eta itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain.
Parker
Joanne L
JL
Cancer Research UK, London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, EN6 3LD, United Kingdom.
Bielen
Aleksandra B
AB
Dikic
Ivan
I
Ulrich
Helle D
HD
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D004247:DNA; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins; C063607:RAD18 protein, S cerevisiae; C471606:RAD6 protein, S cerevisiae; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases