RESUMO
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.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Extratos Celulares , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Adutos de DNA/química , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Técnicas de Inativação de Genes , Humanos , Mutação , Estrutura Terciária de Proteína , Dímeros de Pirimidina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Raios UltravioletaRESUMO
The Rad6/Rad18-dependent monoubiquitination of PCNA plays a crucial role in regulating replication past DNA damage in eukaryotic cells. We show here that in human cell-free extracts, efficient PCNA monoubiquitination requires both the synthesis of relatively long DNA tracts and polymerase idling or stalling at sites of DNA modification or DNA secondary structures. This dual dependency suggests a dynamic process in which, following initiation, the DNA synthesizing complex undergoes modifications that make it competent as a mediator for the activation of the Rad6/Rad18 pathway.
