Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts.
In eukaryotic cells, the Rad6/Rad18-dependent monoubiquitination of the proliferating cell nuclear antigen (PCNA) plays an essential role in the switching between replication and translesion DNA synthesis (TLS). The DNA polymerase Polη binds to PCNA via a consensus C-terminal PCNA-interacting protein (PIP) motif. It also specifically interacts with monoubiquitinated PCNA thanks to a recently identified ubiquitin-binding domain (UBZ). To investigate whether the TLS activity of Polη is always coupled to PCNA monoubiquitination, we monitor the ability of cell-free extracts to perform DNA synthesis across different types of lesions. We observe that a cis-syn cyclobutane thymine dimer (TT-CPD), but not a N-2-acetylaminofluorene-guanine (G-AAF) adduct, is efficiently bypassed in extracts from Rad18-deficient cells, thus demonstrating the existence of a Polη-dependent and Rad18-independent TLS pathway. In addition, by complementing Polη-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Polη activity. The finding that the bypass of a CPD lesion in vitro does not require Ub-PCNA but nevertheless depends on the UBZ domain of Polη, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.
CNRS FRE3211, ESBS, Université de Strasbourg, Bld Sébastien Brandt BP 10413, 67412 Illkirch Cedex, France.
Valérie
V
Schmutz
Régine
R
Janel-Bintz
Jérôme
J
Wagner
Denis
D
Biard
Naoko
N
Shiomi
Robert P
RP
Fuchs
Agnès M
AM
Cordonnier
D002457:Cell Extracts; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D004247:DNA / Q000096:biosynthesis*; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D011740:Pyrimidine Dimers / Q000737:chemistry; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D002457:Cell Extracts; D004247:DNA; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D011740:Pyrimidine Dimers; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C485954:RAD18 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases