Role of the ubiquitin-binding domain of Poleta in Rad18-independent translesion DNA synthesis in human cell extracts.
Fiche publication
Date publication
octobre 2010
Auteurs
Membres identifiés du Cancéropôle Est :
Dr CORDONNIER Agnès
Tous les auteurs :
Schmutz V, Janel-Bintz R, Wagner J, Biard D, Shiomi N, Fuchs RP, Cordonnier AM
Lien Pubmed
Résumé
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 Poleta 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 Poleta 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 Poleta-dependent and Rad18-independent TLS pathway. In addition, by complementing Poleta-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Poleta 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 Poleta, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.
Référence
Nucleic Acids Res. 2010 Oct 1;38(19):6456-65