PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40.
Translesion synthesis (TLS) is a potentially mutagenic method of bypassing DNA damage encountered during replication that requires the recruitment of specialized DNA polymerases to stalled replication forks or postreplicative gaps. Current models suggest that TLS is activated by monoubiquitination of the DNA sliding clamp PCNA. However, in higher organisms, fully effective TLS also requires a noncatalytic function of the Y family polymerase REV1. Using the genetically tractable chicken cell line DT40, we show that TLS at stalled replication forks requires that both the translesion polymerase-interaction domain and ubiquitin-binding domain in the C terminus of REV1 are intact. Surprisingly, however, PCNA ubiquitination is not required to maintain normal fork progression on damaged DNA. Conversely, PCNA ubiquitination is essential for filling postreplicative gaps. Thus, PCNA ubiquitination and REV1 play distinct roles in the coordination of DNA damage bypass that are temporally separated relative to replication fork arrest.
Edmunds
Charlotte E
CE
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Simpson
Laura J
LJ
Sale
Julian E
JE
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000502:physiology*; D004843:Epistasis, Genetic; D006801:Humans; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D016415:Sequence Alignment; D025801:Ubiquitin / Q000378:metabolism*; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D015112:4-Nitroquinoline-1-oxide; D004247:DNA; D009153:Mutagens; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; D018809:Proliferating Cell Nuclear Antigen; D025801:Ubiquitin; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases