Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival.

Luke Springall,Stavros Azinas,Craig D Hughes,Bennett Van Houten,Michelle Simons,Neil M Kad

doi:10.1093/nar/gkx1244

Abstract

Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.

Highlights

Genomes are constantly assaulted by both endogenous and exogenous agents resulting in an array of DNA lesions [1]
Because the DNA tightropes are lifted from the surface, we can be certain that any protein complexes formed are not due to the incidental overlap of proteins/quantum dots (Qdots) that have non-speciically attached to the surface
Nucleotide excision DNA repair is a multi-enzyme process that initially requires damage recognition followed by veriication, incision, removal of the damaged product and inally DNA resynthesis

Summary

Introduction

Genomes are constantly assaulted by both endogenous and exogenous agents resulting in an array of DNA lesions [1]. Solar ultraviolet light (UV) can induce cytotoxic cyclopyrimidine dimers and 6–4 photoproducts [2,3]. Removal of these UV-induced DNA lesions occurs through nucleotide excision repair (NER). This pathway is highly mechanistically conserved from bacteria to mammals and proceeds through the following discrete steps: damage recognition, damage veriication, lesion processing and removal, repair synthesis and ligation. Lesion recognition is achieved by UvrA and UvrB working in concert [4,5,6,7,8,9,10,11,12]. The stage in repair is incision; UvrC is recruited to the UvrB-

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