The Transcription-Repair Coupling Factor Mfd Prevents and Promotes Mutagenesis in a Context-Dependent Manner

Abstract

The mfd (mutation frequency decline) gene was identified by screening an auxotrophic Escherichia coli strain exposed to UV and held in a minimal medium before plating onto rich or minimal agar plates. It was found that, under these conditions, holding cells in minimal (nongrowth) conditions resulted in mutations that enabled cells to grow on minimal media. Using this observation as a starting point, a mutant was isolated that failed to mutate to auxotrophy under the prescribed conditions, and the gene responsible for this phenomenon (mutation frequency decline) was named mfd. Later work revealed that mfd encoded a translocase that recognizes a stalled RNA polymerase (RNAP) at damage sites and binds to the stalled RNAP, recruits the nucleotide excision repair damage recognition complex UvrA₂UvrB to the site, and facilitates damage recognition and repair while dissociating the stalled RNAP from the DNA along with the truncated RNA. Recent single-molecule and genome-wide repair studies have revealed time-resolved features and structural aspects of this transcription-coupled repair (TCR) phenomenon. Interestingly, recent work has shown that in certain bacterial species, mfd also plays roles in recombination, bacterial virulence, and the development of drug resistance.

Keywords: UvrD; excision repair-sequencing (XR-seq); mutation frequency decline (MFD); nucleotide excision repair (NER); transcription-coupled repair (TCR); uvrABC excinuclease.

FIGURE 1

Model for the two nucleotide excision repair pathways in E. coli: general global repair (GGR) and transcription-coupled repair (TCR). UV light induces thymine dimers in DNA which are either directly recognized by UvrA₂B in the GGR pathway or indirectly recognized by RNA polymerase (RNAP) in the TCR pathway. Elongating RNAP stalls when it encounters a dimer in the template strand and recruits the mutation frequency decline (Mfd) translocase, which, in turn, removes RNAP while recruiting UvrA₂B. The two pathways then converge after these initial damage-recognition steps, and UvrA₂ dissociates, leaving a stable preincision complex consisting of UvrB bound to damaged DNA, which now has an altered structure. UvrC is recruited to generate the coupled dual incisions, and UvrD removes UvrC and the damaged oligonucleotide. Repair is completed by synthesis and ligation of the repair patch by DNA polymerase I (PolI) and DNA ligase, respectively.

FIGURE 2

The structure of mutation frequency decline (Mfd). The modular Mfd protein consists of eight domains indicated as boxes in the cartoon (top) and as a rainbow ribbon representation of the crystal structure [PDB ID: 2EYQ (Deaconescu et al., 2006)] viewed with JSmol (bottom). The N-terminus (N) contains the UvrA interaction region (D1a, D2, D1b) which is structurally homologous to the region of UvrB that binds to UvrA. This region of the protein is sequestered in a locked state via interactions with the D7 autoinhibitory domain in the C-terminus (C). Upon interaction with stalled RNAP, the D4 domain, containing the RNA polymerase (RNAP)-interacting domain (RID), binds to the β’ subunit of RNAP which triggers ATP hydrolysis by the helicase motifs in D5-D6 and subsequent DNA translocation and rearrangements ultimately resulting in release of the nascent RNA, removal of RNAP, and recruitment of UvrA₂B repair factors.