A bacterial DNA repair pathway specific to a natural antibiotic

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.

A skipping rope translocation mechanism in a widespread family of DNA repair helicases

“…Our findings do not suggest any specificity of MrfA for mitomycin C-damaged DNA regions. This observation is consistent with the suggestion that the MfrAB helicase/nuclease machinery may be recruited to mitomycin C-induced DNA lesions via a yet to be identified factor ( 10 ).…”

“…by further rolling in of the canopy and/or by lifting of the OB domain via a lid-opening motion about the proline hinge in the CON β-sheet (Figure 7 ; state 1). It is conceivable that such rearrangements are induced when a DNA strand initially docks to the surface of MrfA, upon binding of MrfA to the MrfB exonuclease or upon binding of other factors putatively involved in mitomycin-C damage repair ( 10 ). After DNA loading, the lid may close (Figure 7 ; state 2) and rolling out the canopy allows the OB domain to lock into position at the RecA2 domain.…”

“…Our results reconcile findings from previous functional interrogations of the MrfAB excision repair system. While alteration of MrfA motifs I (K82A), II (S222A) and III (DE185–186AA) suggested that MrfA helicase activity is required for DNA damage repair in vivo , a T134V motif Ib variant still complemented mitomycin C sensitivity ( 10 ). Consistent with these observations, our structural analyses show that in contrast to motif Ib residues G137 and R144, T134 is not directly involved in DNA substrate binding.…”

“…N- and C-terminal MrfA truncations abrogated interaction with the exonuclease MrfB in a bacterial 2-hybrid system ( 10 ). Our structures suggest that such MrfA truncation variants are most likely conformationally compromised; consistent with this notion, our attempts to produce and isolate truncated versions of MrfA that lack either the NTR (MrfA 39–749 ) or the C-terminal CON and MZB domains (MrfA 1–555 ) resulted in insoluble protein.…”

“…For example during one form of NER in bacteria, the DNA damage is first detected by UvrA and verified by the SF2 helicase UvrB; after UvrA release, the UvrC nuclease is recruited and induces nicks upstream and downstream of the damaged site; subsequently, SF1 helicase UvrD and DNA polymerase I remove the post-incision complex, liberate the damaged DNA section and fill the gap, followed strand ligation by DNA ligase ( 9 ). A recent study reported a previously uncharacterized antibiotic-induced DNA excision repair pathway in Bacillus subtilis , that depends on the SF2 ndNTPase, mitomycin repair factor (Mrf) A (formerly YprA), and the metal-dependent exonuclease, MrfB ( 10 ). The MrfAB repair factor complex specifically mends DNA damage induced by mitomycin C, a naturally occurring antibiotic that, upon reductive activation, induces guanosine-specific adducts and causes inter-strand and intra-strand DNA crosslinks ( 11 ).…”