Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart

Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol delta, binds the leading strand upon uncoupling and inhibits TLS. Pol delta is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol delta inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally. In vitro reconstitution of translesion synthesis-mediated replication fork restart shows how DNA Pol eta is recruited to bypass a CPD lesion on the leading strand in the context of the yeast replisome.