Collision with duplex DNA renders Escherichia coli DNA polymerase III holoenzyme susceptible to DNA polymerase IV-mediated polymerase switching on the sliding clamp

Abstract

Organisms possess multiple DNA polymerases (Pols) and use each for a different purpose. One of the five Pols in Escherichia coli, DNA polymerase IV (Pol IV), encoded by the dinB gene, is known to participate in lesion bypass at certain DNA adducts. To understand how cells choose Pols when the replication fork encounters an obstacle on template DNA, the process of polymerase exchange from the primary replicative enzyme DNA polymerase III (Pol III) to Pol IV was studied in vitro. Replicating Pol III forming a tight holoenzyme (Pol III HE) with the sliding clamp was challenged by Pol IV on a primed ssDNA template carrying a short inverted repeat. A rapid and lesion-independent switch from Pol III to Pol IV occurred when Pol III HE encountered a hairpin stem duplex, implying that the loss of Pol III-ssDNA contact induces switching to Pol IV. Supporting this idea, mutant Pol III with an increased affinity for ssDNA was more resistant to Pol IV than wild-type Pol III was. We observed that an exchange between Pol III and Pol IV also occurred when Pol III HE collided with primer/template duplex. Our data suggest that Pol III-ssDNA interaction may modulate the susceptibility of Pol III HE to Pol IV-mediated polymerase exchange.

The SOS system: A complex and tightly regulated response to DNA damage

“…Scotland et al () showed that the switch requires that Pol IV contact not only two discrete sites on the β clamp protein, but Pol III as well. Impaired Pol III–ssDNA interactions (e.g., when polymerase encounters a hairpin‐stem duplex) influence the susceptibility of Pol III HE to Pol IV‐mediated polymerase exchange (Le et al, ). Using single‐molecule time‐lapse microscopy to directly visualize fluorescently labeled pol IV in live cells, Henrikus et al () recently demonstrated that over 90% of foci induced by DNA damage form outside of replisome regions, suggesting that pol IV predominantly carries out non‐replisomal functions, consistent with the postreplicative TLS in gaps behind the replisome model.…”