Cells dedicate tremendous amounts of energy to express essential genes for survival. During transcription, RNA polymerase (RNAP) actively scans the template strand of DNA, stalling when it meets DNA damages. Stalled RNAP prevents repair by the nucleotide excision repair pathway (NER); a sub-pathway of NER named transcription-coupled repair (TCR) resolve this problem by removing RNAP and recruiting repair proteins. In Escherichia coli, a TCR protein named “Mutation Frequency Decline” (Mfd) couples removal of RNAP through its motor activity with recruitment of the NER repair proteins. Mfd can be divided into two functional halves; the N-terminal region (MfdN, domains 1-3) is essential for NER protein recruitment, and the C-terminal region (MfdC, domains 4-7) is responsible for RNAP-interaction and motor activity. Data suggest Mfd undergoes large conformational movement to activate RNAP removal and repair protein recruitment. To study the activation mechanism of Mfd, we created several full-length “hyperactive” mutants by perturbing interactions between MfdN and MfdC. In all mutants, residue 79 in domain 1 is changed from aspartic acid to arginine (D79R), disrupting a key salt bridge interaction with arginine 804 in domain 6. The linker connecting MfdN and MfdC was made cleavable to allow separation of MfdN and MfdC, which enable us to study activities in equal molar concentration. We have studied the effect of the D79R mutation in vivo (cytotoxicity and UV sensitivity) and in vitro (enzyme activity and thermal stability), and demonstrate that this single residues change render the enzyme “hyperactive”. This confirms our model of activation: activation of Mfd results from breaking communication between MfdN and MfdC

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.

The nucleotide excision repair (NER) pathway is essential for repair of UV-induced bulky DNA lesions. NER is divided into two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). UVC radiation has been shown to result in the formation of bulky DNA lesions, which are removed by NER. Previous published reports have shown a role for the p53 tumour suppressor protein in GGR and TCR, but the involvement of p53 in TCR has been controversial. In addition, it has also been suggested that hypoxia affects NER and expression of p53. In the present work, the role of p53, hypoxia and HIF-lα in NER was investigated. It was determined that p53 overexpression in primary human fibroblasts resulted in up-regulation of both the GGR and TCR subpathways of a UV -damaged reporter gene. Pre-treatment of cells with low UVC-fluence and p53 overexpression also induced an upregulation of GGR and TCR. These results are consistent with a p53-dependent upregulation of TCR and GGR of the UVC-damaged reporter gene, as well with a UV-inducible TCR and GGR that is dependent on p53 expression prior to UV treatment. Hypoxia coupled to low pH induced a transient up-regulation of p53 expression and NER in human primary normal fibroblasts and a concomitant decrease in UVC sensitivity. In contrast, in tumour cells hypoxia coupled to low pH resulted in a delayed, but not absent up-regulation of NER, which was p53-independent and did not result in a decrease in UVC sensitivity. We report here that it is the early transient p53-dependent up-regulation induced by hypoxia coupled to acidosis in human primary normal fibroblasts that may play a significant role in cellular UVC sensitivity. These data suggest a different cellular NER response to hypoxia compared to hypoxia coupled to low pH. The NER response to hypoxia and hypoxia coupled with acidosis was also different in primary cells when compared to tumour-derived cells. It was demonstrated that expression of dominant-negative HIF-lα in rat prostate tumour cells results in a reduction in host cell reactivation (HCR) of a UV-damaged reporter gene when compared to that in wild-type HIF-lα cells under normoxic conditions suggesting that basal HIF-lα expression may play an important role in NER. In addition we showed that hypoxia induced an up-regulation of NER in human primary normal fibroblasts that was delayed, but not absent in TCR-deficient CSB cells, suggesting a role for hypoxia in up-regulation of the GGR pathway of NER of a UVdamaged reporter gene. In contrast, HIF-lα-overexpression under conditions of hypoxia resulted in a down-regulation of NER in normal fibroblasts, which was delayed, but not absent in CSB fibroblasts. These results suggest that HIF-1α and CSB are involved in a hypoxia-induced NER response. This work provides further evidence that both GGR and TCR are p53-dependent. In addition, this study provides evidence that hypoxia and hypoxia coupled to acidosis can up-regulate NER in both primary and tumour cells, and that HIF-lα and the CSB protein play an important role in a hypoxia-induced NER response. / Thesis / Doctor of Philosophy (PhD)