DNA damage can potently block the DNA replication machinery. Error prone polymerases can alleviate this block by gaining access to the replisome and synthesizing past the lesion in a process known as translesion synthesis (TLS). Given their low fidelity, the access of TLS polymerases to the replication fork must be tightly regulated in order to minimize error-prone DNA synthesis. Using a combination of in vitro and cell-based single-molecule assays along with biochemical and genetic approaches, we are determining how TLS polymerases are recruited to the bacterial replisome and gain access to the primer template junction. While the TLS polymerase, Pol IV, can simultaneously bind the β<sub>2</sub> processivity clamp with the replicative polymerase, Pol III, in minimal in vitro reconstitutions, we find that it is largely restricted from replisomes in cells in the absence of DNA damage. Interactions between Pol III and the β<sub>2</sub> clamp act as a gatekeeper to minimize TLS polymerase access; strengthening these interactions can dramatically suppress TLS in vitro and in cells. DNA damage dramatically enriches Pol IV near replisomes in cells with previously unknown interactions with the replisome being the major determinant of Pol IV recruitment to stalled forks.

Finding their way: How error-prone polymerases gain access to the bacterial replisome

256th National Meeting (2018)

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