Pol V-Mediated Translesion Synthesis Elicits Localized Untargeted Mutagenesis during Post-replicative Gap Repair

Abstract

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and downstream of a site-specific DNA lesion. Pol V-mediated untargeted mutations are thus spread over several hundred nucleotides, strongly eliciting genetic instability on either side of a given lesion. Consequently, post-replicative gap repair may be a source of untargeted mutations critical for gene diversification in adaptation and evolution.

Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

“…In contrast, repriming of DNA replication downstream of the lesion, leaves a long ssDNA gap (>200 nucleotides) that is repaired primarily by a highfidelity recombination-dependent gap filling mechanism. However, a small fraction of these gaps are filled in by the combined actions of replicative and TLS polymerases in a highly mutagenic manner (TLS at the gap) (Isogawa et al, 2018). This widespread mutagenesis that results during TLS at the gap is more likely to lead to functional genetic alterations compared with the localized mutagenesis that occurs adjacent to the lesion during TLS at the fork.…”

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

“…With respect to error-free bypass of G-AAF by Pol V, TLS is stimulated (~7 fold) in the dnaQ( Q*) strain compared to the mutD5 strain ( Figure 5D), in good agreement with the data observed for Pol V-mediated TLS across TT-CPD and TT(6-4) lesions. Productive TLS, requires Pol V to remain bound to the primer P/T junction in order to synthesize a TLS patch rather than to only insert a single nucleotide directly across from the lesion site Isogawa et al, 2018). As weakening the ε-cleft interaction in cells significantly increased the utilization of TLS over cognate lesions for all three TLS polymerases, we conclude that the ε-cleft contact acts as a gatekeeper to regulate access to the P/T junction.…”

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

“…This gap-suppressing effect of TLS at the fork may play a pivotal role in determining the extent of damage-induced mutagenesis. A recent study has demonstrated that damage-induced mutagenesis is not limited to the lesion site but instead can spread a few hundred nucleotides from the lesion (72). This extended patch of low-fidelity synthesis likely results from gap filling synthesis where bypass of the lesion by the cognate TLS polymerase is followed by the sequential action of multiple polymerases, including the highly mutagenic Pol V, to fill in the remaining ssDNA gap (Fig.…”