Across all domains of life, DNA is replicated by a multi-protein complex known as the replisome. Although the replisome efficiently and accurately replicates undamaged DNA, it can be blocked by unrepaired DNA damage. The DNA damage avoidance pathway translesion synthesis (TLS) allows cells to alleviate this potentially lethal blockade. In TLS, specialized DNA polymerases gain access to the DNA template, bypass the damage, and then relinquish the template to allow the replisome to continue normal DNA synthesis. Because TLS polymerases are error-prone, however, their access to the template must be tightly regulated to avoid harmful mutations. Previously we showed that the most abundant TLS polymerase in the model bacterial species <i>Escherichia coli</i>, Pol IV, is not strongly enriched near sites of DNA replication in normally growing cells. Instead, Pol IV is recruited in response to DNA damage, in part through interactions with the replication processivity factor β clamp. These results suggest a mechanism by which the cell avoids unnecessary access of Pol IV to the template, but they raise other questions. In particular, it remains unclear what role the two distinct interactions between Pol IV and β play in the recruitment process, whether interactions with other proteins are important for recruitment, and what molecular-level signals lead to Pol IV recruitment. Using particle-tracking photoactivation localization microscopy (PALM), we visualize the behavior of single Pol IV molecules in live <i>E. coli</i> cells. We elucidate the role of the two distinct Pol IV-β contacts and we show that an interaction with single-stranded DNA-binding protein (SSB) plays the major role in Pol IV recruitment. Finally, we provide new insight into what molecular events trigger Pol IV recruitment. These results provide a possible model for the recruitment of other proteins that respond to DNA damage and to other replication perturbations in cells.

Elucidating the molecular mechanisms of translesion polymerase recruitment

ACS Fall 2020 Virtual Meeting

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