Role of the ubiquitin-binding domain of Pol eta 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 Pol eta 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 Pol eta 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 Pol eta-dependent and Rad18-independent TLS pathway. In addition, by complementing Pol eta-deficient cells with PIP and UBZ mutants, we show that each of these domains contributes to Pol eta 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 Pol eta, reveals that this domain may play a novel role in the TLS process that is not related to the monoubiquitination status of PCNA.