Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria.

Asha Mary Joseph,Saheli Daw,Anjana Badrinarayanan,Ismath Sadhir

doi:10.7554/elife.67552

Abstract

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

Highlights

DNA damage is a threat to genome integrity and can lead to perturbations to processes of replication and transcription
Given that DNA damage can occur in any cell type whether actively replicating or not, coordinated activity of Nucleotide Excision Repair (NER) and low-fidelity polymerases can serve as a potential mechanism through which non-replicating cells such as bacteria in stationary phase or cells in other differentiated phases increase their chances of survival under damage
Apart from -clamp foci, we found that the replicative polymerase, DnaE, was unable to dissociate during damage recovery in cells lacking dnaE2 (Figure 4C), suggesting that the replicative polymerase alone cannot complete synthesis across these NER-generated single-stranded DNA (ssDNA) tracts

Summary

Introduction

DNA damage is a threat to genome integrity and can lead to perturbations to processes of replication and transcription. Coli, where activities of PolIV and PolV are well-studied, in vivo investigations of DnaE2 function in damage tolerance and cellular survival are limited This becomes important, given the emerging evidences across domains of life ascribing diverse functions to these lowfidelity polymerases beyond their canonical function of replication-associated lesion bypass (Joseph & Badrinarayanan, 2020). DnaE2 functions in gap-filling damaged DNA in non-replicating cells This is facilitated by de novo loading of replisome components (SSB, HolB (part of the clamp loader complex), β-clamp and replicative polymerase) at long ssDNA gaps likely generated by a subset of NER events. We find that this form of gap-filling in non-replicating cells promotes cell cycle restoration and cell division, upon release into replication-permissive conditions. Given that DNA damage can occur in any cell type whether actively replicating or not, coordinated activity of NER and low-fidelity polymerases can serve as a potential mechanism through which non-replicating cells such as bacteria in stationary phase or cells in other differentiated phases increase their chances of survival under damage

Results

Discussion

Materials and methods

Declaration of interests