Abstract
All organisms possess DNA repair pathways that are used to maintain the integrity of their genetic material. Although many DNA repair pathways are well understood, new pathways continue to be discovered. Here, we report an antibiotic specific DNA repair pathway in Bacillus subtilis that is composed of a previously uncharacterized helicase (mrfA) and exonuclease (mrfB). Deletion of mrfA and mrfB results in sensitivity to the DNA damaging agent mitomycin C, but not to any other type of DNA damage tested. We show that MrfAB function independent of canonical nucleotide excision repair, forming a novel excision repair pathway. We demonstrate that MrfB is a metal-dependent exonuclease and that the N-terminus of MrfB is required for interaction with MrfA. We determined that MrfAB failed to unhook interstrand cross-links in vivo, suggesting that MrfAB are specific to the monoadduct or the intrastrand cross-link. A phylogenetic analysis uncovered MrfAB homologs in diverse bacterial phyla, and cross-complementation indicates that MrfAB function is conserved in closely related species. B. subtilis is a soil dwelling organism and mitomycin C is a natural antibiotic produced by the soil bacterium Streptomyces lavendulae. The specificity of MrfAB suggests that these proteins are an adaptation to environments with mitomycin producing bacteria.
Highlights
A defining feature of biology is the ability to reproduce, which requires replication of the genetic material
MrfAB are a putative helicase and exonuclease, respectively, and we demonstrate that conserved residues required for their activities are important for function in vivo
We monitored DNA repair status over time using RecA-GFP as a reporter, and we show that deletion of mrfAB and uvrABC results in a synergistic decrease in RecA-GFP foci, suggesting that MrfAB are part of a novel nucleotide excision repair pathway in bacteria
Summary
A defining feature of biology is the ability to reproduce, which requires replication of the genetic material. High fidelity DNA replication depends on the integrity of the template DNA which can be damaged by UV light, ionizing radiation, and numerous chemicals (Friedberg et al, 2006). Many DNA damaging agents have been used as chemotherapeutics and are produced from natural sources such as bacteria, fungi, or plants (Demain & Vaishnav, 2011). One such naturally produced antibiotic is mitomycin C (MMC), originally isolated from Streptomyces lavendulae (Hata et al, 1956). MMC is produced as an inactive metabolite that must be activated by enzymatic or chemical reduction to react with DNA (Tomasz, 1995). MMC reacts with guanine residues in DNA and results in three principle modifications
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