Beyond the Lesion: Back to High Fidelity DNA Synthesis.

High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine.

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves 'on-the-fly' translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.