Mfd Gene Research Articles

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis

Invitro and invivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E.coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

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

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.

Lethal Mechanisms of Nostoc-Synthesized Silver Nanoparticles Against Different Pathogenic Bacteria.

BackgroundIncreasing antibiotic resistance and the emergence of multidrug-resistant (MDR) pathogens have led to the need to develop new therapeutic agents to tackle microbial infections. Nano-antibiotics are a novel generation of nanomaterials with significant antimicrobial activities that target bacterial defense systems including biofilm formation, membrane permeability, and virulence activity.PurposeIn addition to AgNO3, the current study aimed to explore for first time the antibacterial potential of silver nanoparticles synthesized by Nostoc sp. Bahar_M (N-SNPs) and their killing mechanisms against Streptococcus mutans, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium.MethodsPotential mechanisms of action of both silver species against bacteria were systematically explored using agar well diffusion, enzyme (lactate dehydrogenase (LDH) and ATPase) and antioxidant (glutathione peroxidase and catalase) assays, and morphological examinations. qRT-PCR and SDS-PAGE were employed to investigate the effect of both treatments on mfD, flu, and hly gene expression and protein patterns, respectively.ResultsN-SNPs exhibited greater biocidal activity than AgNO3 against the four tested bacteria. E. coli treated with N-SNPs showed significant surges in LDH levels, imbalances in other antioxidant and enzyme activities, and marked morphological changes, including cell membrane disruption and cytoplasmic dissolution. N-SNPs caused more significant upregulation of mfD expression and downregulation of both flu and hly expression and increased protein denaturation compared with AgNO3.ConclusionN-SNPs exhibited significant inhibitory potential against E. coli by direct interfering with bacterial cellular structures and/or enhancing oxidative stress, indicating their potential for use as an alternative antimicrobial agent. However, the potential of N-SNPs to be usable and biocompatible antibacterial drug will evaluate by their toxicity against normal cells.

Single Molecule Studies of Bacterial Transcription Coupled Repair

Transcription coupled repair (TCR), a sub-pathway of the nucleotide excision repair mechanism, is activated when a RNA polymerase (RNAP) is arrested during transcription by DNA damage. TCR is a ubiquitous cellular response important for maintenance of DNA integrity. Some human genetic disorders are associated with defect on TCR, like the Cockayne syndrome for instance. In the bacterium Escherichia coli, TRCF, the product of the mfd gene, is the DNA translocase that couples transcription and DNA repair: it recognizes a stalled ternary elongation complex, dissociates it, and recruits the UvrABC repair machinery.

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Transcription-repair coupling factor (TRCF; the product of the mfd gene) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNA polymerase stalled at a noncoding lesion in the DNA template strand, uses the energy from ATP hydrolysis to disrupt the transcription complex, and stimulates DNA repair by recruiting UvrA, a component of the nucleotide excision repair machinery, to the site. TRCF is a large (130 kDa) multifunctional protein with a complex structure–function relationship consisting of a compact arrangement of eight structured domains linked by flexible linkers. Through a conserved, intramolecular, interdomain interaction, TRCF is held in a conformation in which its enzymatic activities (ATPase activity and DNA translocase activity) are strongly repressed. Disruption of the repressive interdomain interaction by amino acid substitutions within the interface derepresses ATPase and DNA translocase activities. In this work, we have shown that derepressed TRCF mutants are dramatically sensitized to limited proteolysis compared with repressed TRCF, pointing to an altered conformational state. Analysis of the protease cleavage sites mapped onto the structure of the repressed TRCF conformation indicates that (1) the cleavage sites tend to cluster at linkers connecting the TRCF structured domains, and (2) many of the cleavage sites sensitized in the derepressed TRCF are partially or completely buried to protease access in the repressed TRCF structure. We conclude that TRCF derepression is associated with profound conformational changes that primarily involve a reorganization of the interdomain interactions.

Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction

The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.

Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), α-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3 → Arg + revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 ( alkB −) strain in comparison with the AB1157 ( alkB +) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd +, and no repair in BS87 mfd − cells. Determination of specificity of Arg + revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT → TA transversions by supL suppressor formation (1meA) and GC → AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB − mfd − bacteria but not in the alkB + mfd − counterpart.

The Helicobacter pylori Mfd protein is important for antibiotic resistance and DNA repair

A Helicobacter pylori mutant defective in a putative mfd gene was constructed and characterized. The mfd gene is required for DNA repair and is involved in DNA recombination processes. The mfd mutant strain displayed a greatly increased susceptibility to antibiotics, indicating that this gene plays a significant role in the antibiotic resistance of H. pylori strain J99.

Key Role of Mfd in the Development of Fluoroquinolone Resistance in Campylobacter jejuni

Campylobacter jejuni is a major food-borne pathogen and a common causative agent of human enterocolitis. Fluoroquinolones are a key class of antibiotics prescribed for clinical treatment of enteric infections including campylobacteriosis, but fluoroquinolone-resistant Campylobacter readily emerges under the antibiotic selection pressure. To understand the mechanisms involved in the development of fluoroquinolone-resistant Campylobacter, we compared the gene expression profiles of C. jejuni in the presence and absence of ciprofloxacin using DNA microarray. Our analysis revealed that multiple genes showed significant changes in expression in the presence of a suprainhibitory concentration of ciprofloxacin. Most importantly, ciprofloxacin induced the expression of mfd, which encodes a transcription-repair coupling factor involved in strand-specific DNA repair. Mutation of the mfd gene resulted in an approximately 100-fold reduction in the rate of spontaneous mutation to ciprofloxacin resistance, while overexpression of mfd elevated the mutation frequency. In addition, loss of mfd in C. jejuni significantly reduced the development of fluoroquinolone-resistant Campylobacter in culture media or chickens treated with fluoroquinolones. These findings indicate that Mfd is important for the development of fluoroquinolone resistance in Campylobacter, reveal a previously unrecognized function of Mfd in promoting mutation frequencies, and identify a potential molecular target for reducing the emergence of fluoroquinolone-resistant Campylobacter.

Transcription coupled nucleotide excision repair in Escherichia coli can be affected by changing the arginine at position 529 of the β subunit of RNA polymerase

The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the β subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the β subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR.

Structural Basis for Bacterial Transcription-Coupled DNA Repair

Coupling of transcription and DNA repair in bacteria is mediated by transcription-repair coupling factor (TRCF, the product of the mfd gene), which removes transcription elongation complexes stalled at DNA lesions and recruits the nucleotide excision repair machinery to the site. Here we describe the 3.2 A-resolution X-ray crystal structure of Escherichia coli TRCF. The structure consists of a compact arrangement of eight domains, including a translocation module similar to the SF2 ATPase RecG, and a region of structural similarity to UvrB. Biochemical and genetic experiments establish that another domain with structural similarity to the Tudor-like domain of the transcription elongation factor NusG plays a critical role in TRCF/RNA polymerase interactions. Comparison with the translocation module of RecG as well as other structural features indicate that TRCF function involves large-scale conformational changes. These data, along with a structural model for the interaction of TRCF with the transcription elongation complex, provide mechanistic insights into TRCF function.

Crystallization and preliminary structure determination ofEscherichia coliMfd, the transcription-repair coupling factor

Transcription-repair coupling factors (TRCFs) are SF2 ATPases that couple transcription to DNA-damage repair by recognizing and removing RNA polymerase-elongation complexes stalled at DNA lesions and recruiting the nucleotide excision-repair machinery to the damaged sites. As a first step towards understanding the TRCF mechanism, the 130 kDa Escherichia coli TRCF (the product of the mfd gene) has been overexpressed, purified and crystallized using an unusual precipitant, pentaerythritol ethoxylate. Initial phases were obtained using single-wavelength anomalous dispersion with a highly redundant 4 A resolution data set collected from selenomethionyl-substituted crystals and dramatically improved by density modification and phase extension to 3.2 A resolution. Model building and refinement, which are in progress, will provide insight into transcription-coupled DNA-repair pathways, as this represents the first TRCF to be crystallized to date.

Characterisation of the Escherichia coli mfd promoter.

The bacterial mfd gene encodes a transcription-repair coupling factor that mediates the preferential repair of DNA damage in the template strand of active transcriptional units. In this report, the transcription start site for the Escherichia coli mfd gene was determined in vivo and in vitro, and the DNA determinants for mfd transcription by deletion and site-directed mutagenesis were defined. A canonical sigma(70)-dependent promoter, mfd P1, was responsible for the majority of mfd transcription, and a core region consisting of residues -42 to +5 was sufficient for full activity in rich and minimal media.

Characterization of Transcription-Repair Coupling Factors in E. Coli and Humans

This chapter discusses the characterization of transcription-repair coupling factors in Escherichia coli (E. Coli ) and humans. Transcription repair coupling required a functional nucleotide excision-repair pathway and transcription by E. coli RNAP; using this in vitro assay, a protein named transcription-repair coupling factor (TRCF) was purified, and the gene encoding TRCF was identified as the mutation frequency decline (mfd) gene responsible for MFD in vivo. In E. coli, the TRCF, also called the “Mfd protein”, recognizes the stalled polymerase and performs two functions. First, it dissociates the stalled polymerase and nascent RNA from the template. Then, as a consequence of its affinity for the nucleotide excision repair enzyme (A) BC excinuclease, it delivers the repair enzyme to the damage site. DNA duplexes of known sequences and lengths containing promoters, defined DNA damage at a unique site, and a radiolabel were generated to study the mechanism of TRCF protein action. Two such substrates, each 138 bp in length are discussed. Complementation analysis indicates that defects in one of two proteins, CSA and CSB, are associated with the Cockayne's syndrome. The comparisons between the E. coli TRCF and human CSB proteins are tabulated.

Human Transcription-Repair Coupling Factor CSB/ERCC6 Is a DNA-stimulated ATPase but Is Not a Helicase and Does Not Disrupt the Ternary Transcription Complex of Stalled RNA Polymerase II

Transcription is coupled to repair in Escherichia coli and in humans. Proteins encoded by the mfd gene in E. coli and by the ERCC6/CSB gene in humans, both of which possess the so-called helicase motifs, are required for the coupling reaction. It has been shown that the Mfd protein is an ATPase but not a helicase and accomplishes coupling, in part, by disrupting the ternary complex of E. coli RNA polymerase stalled at the site of DNA damage. In this study we overproduced the human CSB protein using the baculovirus vector and purified and characterized the recombinant protein. CSB has an ATPase activity that is stimulated strongly by DNA; however, it neither acts as a helicase nor does it dissociate stalled RNA polymerase II, suggesting a coupling mechanism in humans different from that in prokaryotes. CSB is a DNA-binding protein, and it also binds to XPA, TFIIH, and the p34 subunit of TFIIE. These interactions are likely to play a role in recruiting repair proteins to ternary complexes formed at damage sites.

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Structure and Function of Transcription-Repair Coupling Factor: I. STRUCTURAL DOMAINS AND BINDING PROPERTIES

The 130-kDa mfd gene product is required for coupling transcription to repair in Escherichia coli. Mfd displaces E. coli RNA polymerase (Pol) stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription. Here, the interactions of Mfd (transcription-repair coupling factor, TRCF) with DNA, RNA Pol, and UvrA were investigated. TRCF bound nonspecifically to double stranded DNA; binding to DNA produced alternating DNase I-protected and -hypersensitive regions, suggesting possible wrapping of the DNA around the enzyme. Weaker binding to single stranded DNA and no binding to single stranded RNA were observed. DNA binding required ATP, and hydrolysis of ATP promoted dissociation. Removal of a stalled RNA Pol also requires ATP hydrolysis. Apparently, TRCF recognizes a stalled elongation complex by directly interacting with RNA Pol, since binding to a synthetic transcription bubble was no stronger than binding to double stranded DNA, and binding to free RNA Pol holoenzyme and to initiation and elongation complexes in the absence of adenosine 5'-O-(thiotriphosphate) were observed. Structure-function analysis showed that residues 379-571 are involved in binding to a stalled RNAP. The helicase motifs region, residues 571-931, binds to ATP and duplex polynucleotide (DNA:DNA or DNA:RNA). Dissociation of the ternary complex upon hydrolysis of ATP also requires the carboxyl terminus of TRCF. Finally, residues 1-378 bind to UvrA and deliver the damage recognition component of the excision nuclease to the lesion.

NUCLEOTIDE EXCISION REPAIR

Nucleotide excision repair is the major DNA repair mechanism in all species tested. This repair system is the sole mechanism for removing bulky adducts from DNA, but it repairs essentially all DNA lesions, and thus, in addition to its main function, it plays a back-up role for other repair systems. In both pro- and eukaryotes nucleotide excision is accomplished by a multisubunit ATP-dependent nuclease. The excision nuclease of prokaryotes incises the eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified nucleotide and thus excises a 12-13-mer. The excision nuclease of eukaryotes incises the 22nd, 23rd, or 24th phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion and thus removes the adduct in a 27-29-mer. A transcription repair coupling factor encoded by the mfd gene in Escherichia coli and the ERCC6 gene in humans directs the excision nuclease to RNA polymerase stalled at a lesion in the transcribed strand and thus ensures preferential repair of this strand compared to the nontranscribed strand.

Molecular Mechanism of Transcription-Repair Coupling

Lesions in the transcribed strand block transcription and are repaired more rapidly than lesions in the nontranscribed (coding) strand which do not block RNA polymerase (RNAP). It has been shown previously that in Escherichia coli the mfd (mutation frequency decline) gene is necessary for strand-specific repair. The mfd gene was cloned and sequenced and the Mfd protein was purified and used to reconstitute strand-specific repair in a completely defined system. The mfd gene encodes a protein of 130 kilodaltons and contains the so-called "helicase motifs," a leucine zipper motif, and regions of sequence similarity to UvrB and RecG proteins. The Mfd protein was shown to (i) displace RNAP stalled at a lesion in an adenosine triphosphate-dependent reaction, (ii) bind to the damage recognition subunit (UvrA) of the excision nuclease, and (iii) stimulate the repair of the transcribed strand only when transcription is taking place. Thus, Mfd appears to target the transcribed strand for repair by recognizing a stalled RNAP and actively recruiting the repair enzyme to the transcription blocking lesion as it dissociates the stalled RNAP.

Excision repair at individual bases of the Escherichia coli lacI gene: relation to mutation hot spots and transcription coupling activity.

To determine whether base-to-base variations in the rate of excision repair influence the distribution of mutations, we have developed a method to measure UV photoproducts at individual nucleotides in the Escherichia coli chromosome. Specific gene fragments are 3' end-labeled using a sequence-specific oligonucleotide to direct the site of labeling, and photoproducts are identified by enzymatic incision. On the nontranscribed strand of the E. coli lacI gene, the cyclobutane pyrimidine dimer frequency was 2- to 8-fold higher in chromosomal DNA than in a cloned DNA fragment. The chromosomal lesion frequency corresponded to the frequency of UV-induced mutations at mutation hot spots reported in the literature. Only 0-30% of cyclobutane dimers at various sites on this strand were excised in 20 min. In contrast, repair on the transcribed strand was 80-90% complete in 20 min. However, the transcribed strand contained an excision repair "slow spot" at the site of its single mutation hot spot: At this site, no repair occurred for the first 10 min, after which repair proceeded more slowly than typical of that strand. In an mfd strain, deficient in a factor that couples repair to transcription in cell extracts, the excision rate at individual nucleotides on the transcribed strand was minimal at most sites for at least 30 min. Wild-type E. coli's bias for producing mutations at photoproducts on the nontranscribed strand, reported to require the mfd gene, therefore appears to be due to an excision repair system specific for the transcribed strand of chromosomal DNA.