| dc.description.abstract | Introduction
Nucleotide excision repair (NER) is a highly conserved pathway involved in repair of a wide variety of structurally unrelated DNA lesions. One of the well characterized NER systems is from E. coli which involves UvrABC nucleases. NER consists of two related sub-pathways: global genomic repair (GGR), which removes lesions from the overall genome, and transcription coupled repair (TCR), which removes lesions from the transcribed strand of active genes. Bulky DNA lesions such as cyclobutane pyrimidine photodimers (CPD) induced by UV irradiation block RNA polymerase (RNAP) during transcription. In bacteria, a gene product of mfd called transcription repair coupling factor (TRCF) or Mfd is required for TCR. Bacterial Mfd interacts with the stalled RNAP, displaces it from the DNA and recruits NER proteins at the site of damage. Mfd, thus contributes to the faster repair of the transcribed strand compared to the non-transcribed strand for similar kind of lesions.
Intracellular pathogens like M. tuberculosis are constantly exposed to a variety of stress conditions inside the host, mainly due to host defense systems and antibiotic treatments. It is therefore, extremely important for bacteria to have DNA damage repair and reversal mechanisms that can efficiently counteract these effects. However, very little is known about DNA repair systems in M. tuberculosis compared to other bacteria. Sequencing of M. tuberculosis genome revealed the presence of NER associated genes including a putative mfd. Additionally, due to the high GC content of genome as well as the DNA damage prone host environment, the transcription in M. tuberculosis may encounter the problems, which are not apparent in other bacteria. Therefore, the gene like mfd may play very important role in physiology of M. tuberculosis. In the present study, we describe the biochemical and functional characterization of Mfd from M. tuberculosis (MtbMfd) and discuss its unusual properties.
Biochemical characterization of MtbMfd
Genome analysis of M. tuberculosis as well as the sequence alignment studies revealed that MtbMfd is 1234 amino acids long multifunctional protein having various domains specialized for different functions. Cloning of Mtbmfd was carried out by reconstructing the full length gene from three PCR amplified fragments using genomic DNA as a template. Complementation study using Mtbmfd suggested that the gene of interest complements E. coli counterpart and increases survival of UV irradiated cells. To further characterize the function of Mtbmfd, a road block reporter assay was performed, which indicates that the MtbMfd interacts with stalled E. coli RNAP and displaces it from the site of transcription resulting in low reporter gene activity. The MtbMfd protein was expressed and purified by using various chromatographic techniques, and confirmed by mass spectrometry. In addition to full length protein, a number of truncated MtbMfd constructs were generated and purified to homogeneity. Mfd is a motor protein and requires ATP hydrolysis in order to translocate along DNA. The signature motifs of superfamily 2 helicases / ATPases are present at the C-terminal of Mfd along with translocase motif which is highly homologous to motif present in RecG helicase. To analyze the kinetics of ATP hydrolysis of MtbMfd and its truncated proteins, ATPase reactions were carried out using γ32P-ATP as a tracer. Wild-type MtbMfd exhibited ATPase activity, which was stimulated ~1.5 fold in presence of dsDNA. The mutant MtbMfd (D778A), which harbors mutation in one of the key residues of Walker B motif of the ATPase domain showed negligible ATPase activity indicating the importance of residue D778 for ATP hydrolysis. While the C-terminal domain (CTD) comprising amino acids 600 to 1234 showed elevated ATPase activity, the N-terminal domain (NTD) containing the first 500 amino acid residues was able to bind ATP but deficient in hydrolysis. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ~10-fold compared to full-length MtbMfd. The translocase activity of MtbMfd was measured by an oligonucleotide displacement assay and it was found that full length MtbMfd and CTD have a very weak translocase activity whereas, MfdΔC exhibited efficient translocation along DNA in ATP dependent manner. These results provide a direct correlation between translocase and ATPase activity of MtbMfd, and suggest possibly an auto-regulatory function for the extreme C-terminus of MtbMfd. Oligomeric status of MtbMfd was determined using various techniques including gel filtration chromatography and it was found that MtbMfd exists as monomer and hexamer in solution. The monomer showed increased ATPase activity and susceptibility to proteases compared to the hexameric form. MfdΔC, on the other hand, was predominantly monomer in solution implicating importance of the extreme C-terminal region in oligomerization of protein. Taken together, the biochemical evidence suggests that monomeric MtbMfd is an active form and oligomerization provides stability to the protein. One important finding of the present study is the binding of ATP to NTD of MtbMfd. All Mfd NTDs resemble UvrB and possesses the degenerate ATPase motifs. Indeed, on the basis of sequence and structural similarities, it has been suggested that Mfds have evolved from UvrB incorporating an additional translocase activity. UvrB has a cryptic ATPase activity while the NTD of Mfd may have lost the activity as it possesses degenerate Walker motifs. In contrast, NTD of MtbMfd binds ATP but is hydrolysis deficient. A closer comparison of the amino acid sequences in the Walker A motif reveal that conserved K 45 of UvrB has been replaced by R in case of NTD of MtbMfd. It has been shown previously that mutation of K 45 to A, D and R led to a loss of ATPase activity of UvrB. Thus, MtbMfd seems to be a natural mutant of UvrB. Since NTD harbors an intact UvrA interacting domain, when it is expressed it may sequester the cellular pool of UvrA leading to dominant negative phenotype. When UV survival assays were carried out, cells expressing NTD showed hyper-sensitivity to UV light – a typical characteristic of NER deficiency. In addition, in vitro NER assay clearly suggested that NTD sequesters pool of UvrA inside the cell and blocks both GGR and TCR which further affects the mutation frequency of bacterial cells.
Influence of MtbMfd on elongation state of RNAP
The movement of RNAP along the template during transcription elongation is not uniform and is interrupted due to various factors. To overcome transcription elongation interruptions, a number of proteins viz. Mfd, Gre and
Nus act on RNAP and modify its activity. RNAP displacement and transcript release experiments showed that MtbMfd influenced the elongating RNAP by more than one way. MtbMfd displaced stalled RNAP, which was blocked by NTP starvation on T7A1 promoter based template in a concentration and time-dependent manner. RNAP displacement activity of MtbMfd was shown to depend on ATP or dATP hydrolysis. On the other hand nucleotides like ADP, GTP, CTP and ATPγS did not support the RNAP displacement activity. However, in presence of ATPγS, MtbMfd was able to bind stalled complex but unable to displace RNAP suggesting that ATP or dATP hydrolysis is important for MtbMfd function. On the other hand, MtbMfd did not affect initiating RNAP when σ factor was still bound suggesting that upstream DNA is necessary for Mfd function. To assay RNA or transcript release activity of MtbMfd after transcription complex disruption, immobilized transcription complex assay was carried out. Immobilized stalled complex was generated by UTP and CTP starvation on biotinylated T7A1 promoter based template which can be affixed to temporary pellet in presence of streptavidin beads. It was found that MtbMfd released RNA into a supernatant fraction in a concentration-dependent manner suggesting that MtbMfd releases transcript after ternary complex disruption. MtbMfd released transcript in an energy-dependent manner and both ATP and dATP supported the activity, which allows the complete separation of RNA release from RNA synthesis inside the cell. An ATPase mutant of MtbMfd (MfdD778A) failed to release transcript, which further supported that ATP hydrolysis is important for MtbMfd function. Since both Mfds and RNAPs are evolutionary conserved proteins, to analyze the effect of MtbMfd on other bacterial RNAPs, displacement and release assays were carried out. Stalled complexes were generated using EcoRNAP (E. coli), MsRNAP (M. smegmatis) and MtbRNAP (M. tuberculosis) on T7A1 promoter based template. It was observed that MtbMfd was able to displace all the three RNAPs from stalled elongation complex as well as released transcript with varying efficiency. MtbMfd showed optimal displacement and release activity in presence of mycobacterial RNAPs.
Transcription elongation complexes adopt various conformations and exist as different isomerized states during elongation. In an active elongation complex
the 3'-OH polymerizing end of transcript aligns with an active centre of the RNAP. However, one of the most common and intrinsic properties of RNAP is backtracking or reverse translocation, which leads to misalignment of 3'-OH polymerizing end from an active centre of the polymerase. It is of interest to know if backtracking affects MtbMfd function. It is likely that complexes blocked by lesions inside the cell might tend to backtrack, and different translocational isomers possibly have different sensitivities to MtbMfd action which may illuminate the overall mechanism of MtbMfd. Backtracking of RNAP was induced on +20 and +39 stalled complexes and the effect of MtbMfd was analyzed in presence of NTPs in the reaction. It was found that arrested or backtracked complexes were restored to the forward position by the activity of MtbMfd in presence of NTP resulting into productive elongation. These results suggest that arrested RNAP again resumes transcription if conditions are favorable; otherwise, MtbMfd further assists RNAP to dissociate which leads to release of transcript.
Anti-backtracking activity of MtbMfd might have important function in cellular metabolism and it has been speculated that Mfd could play more general role during transcription apart from repair. To explore the role of MtbMfd as a transcription factor and effect of MtbMtb on transcription processes in the mycobacteria, a variety of T7A1 promoter based templates were generated. These templates were derived from genes of M. tuberculosis and E. coli having varying GC content (39-81 %). The rationale behind this experiment is that the high GC content of mycobacteria and the template derived from mycobacterial genes may pose as sequence dependent structural constraints and hence block the RNAP during transcription. By anti-backtracking activity of MtbMfd these paused complexes may get relieved, leading to efficient transcription by RNAP which may lead to the formation of more full length transcript. To analyze the effect of MtbMfd, purified templates of different GC content were incubated with RNAP and MtbMfd to carry out in vitro transcription. Although, in case of multiple rounds of transcription, multiple pauses were observed even in presence of MtbMfd. However, in presence MtbMfd around 1.5 - 2 fold increased full-length transcripts were observed suggesting that MtbMfd assisted RNAP during elongation to overcome sequence dependent pause. To avoid multiple pauses that are likely to occur due to the initiation of multiple round of transcription, and trailing effect of RNAP itself, single round of transcriptions were carried out in presence of heparin. Sequence specific pauses were observed with increasing GC percentage in template suggesting that indeed high GC content contributes to transcription pause. At the same time, MtbMfd in the reaction increased the amount of full length transcript by 1.5 - 2.0 fold probably by pushing paused RNAP forward to resume elongation.
Taken together, this study investigates the biochemical properties of MtbMfd and its mechanism of action. In addition, it explores the importance of the coupling of transcription to repair in M. tuberculosis as well as the overall proof reading mechanism of transcription elongation in the GC rich genome of mycobacteria. | en_US |
