DNA repair proteins in mycobacteria and their physiological importance Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated. I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role. Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins. II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed. III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV. Physiological importance of MsmUdgX in M. smegmatis MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process.

Maintenance of a topological homeostasis by introduction and removal of the supercoils to relieve excessive strain on the DNA is a hallmark of topoisomerase function in the cell. The requirement of the topoisomerases during DNA transaction processes marks a ubiquitous presence of the enzymes in all the life forms. Different reactions carried out by the enzymes include relaxation of positive and negative supercoils required majorly during DNA replication and transcription, decatenation at the end of DNA replication to separate the daughter chromosomes and removal of lethal knots generated in the circular chromosome. In eubacteria, the enzymes introduce negative supercoils to facilitate easier strand separation for DNA transaction processes. However, in thermophiles, a different enzyme maintains the genome in a positively supercoiled form to protect from denaturation by excessive heat. These varied functions are carried out by different topoisomerases. Therefore, each organism maintains a minimum required set of the enzymes and the absence of a certain enzyme may be compensated for by topoisomerases with dual functions. For example, Mycobacterium tuberculosis and many other slow growing mycobacteria do not possess topoisomerase IV or its homologs. In these organisms, the DNA gyrase is suggested to carry out both negative supercoiling and decatenation reactions. Therefore, the mycobacterial DNA gyrase must be able to manage between both the functions in vivo. In contrast, Mycobacterium smegmatis and few other mycobacteria contain an additional type II topoisomerase which does not resemble any known type II enzyme but could catalyze relaxation and decatenation reactions. Importantly, the enzyme displays a unique ability to introduce limited positive supercoils and may have certain functions inside the cell which remains to be studied. Owing to the indispensability for bacterial survival topoisomerases present themselves as important drug targets. A large number of inhibitors have been found to inhibit the enzyme and thereby killing the bacterial. Among these, quinolones are successfully being used as broad spectrum antibacterial drugs. Although the commonly used quinolones inhibit many bacterial pathogens, a reduced susceptibility is exhibited by some of the pathogens e.g. Mycobacterium tuberculosis. To circumvent the lower efficacy of existing drugs, new and modified quinolones have been developed which are highly effective against mycobacteria. The difference in the susceptibility may be conferred by a difference in the chemical property of the drug and the interacting residues present in the enzyme. In the present thesis efforts have been made to understand the mechanism of the type II topoisomerases from mycobacteria and drug action on these enzymes. The thesis is divided into four chapters. In Chapter I of the thesis an introduction is provided on the topoisomerases, their classification and different reactions catalyzed by these enzymes. As the work in present thesis has been carried out with type II topoisomerases, introduction of type II enzymes, their structure and mechanisms is elaborated. DNA gyrase, its mechanism of reaction and in vitro and in vivo functions are explained in great detail. DNA gyrase and topoisomerase IV are targeted by a range of different inhibitors. These different classes of inhibitors and their mechanism of action are described. Finally, the mechanism of mycobacterial DNA gyrase with structural information and the current understanding of quinolone action on the enzyme are explained. The chapter ends with the objective of the study in the present thesis. In chapter II, the studies are aimed at understanding the molecular basis for decatenation carried out by mycobacterial DNA gyrase. Previous work from the laboratory showed that the enzyme can carry out decatenation more efficiently than its homolog from E. coli. It was shown that the mycobacterial enzyme binds two DNA molecules in trans in a length dependent manner. The ability to bind the second DNA is conferred upon the holoenzyme by ATPase subunit (GyrB) subunit which alone can bind DNA. Similar studies using topo IV from E. coli, the strongest known decatenase showed binding of two DNA molecules and the second DNA binding by ATPase (ParE) subunit. However, GyrB subunit from E. coli DNA gyrase, a weaker decatenase, does not bind second DNA molecule efficiently. The results provide a general mechanism for decatenation by type II enzymes in which efficient binding of second DNA is important. In Chapter III, studies have been carried out using topo NM, an atypical type II topoisomerase from Mycobacterium smegmatis. The enzyme has been characterized previously in the laboratory. In addition to efficient decatenation and relaxation, the enzyme exhibits a unique ability to introduce positive supercoils into the DNA. As demonstrated for the mycobacterial DNA gyrase and topo IV in the Chapter II, the ATPase subunit (Topo N) of topo NM, binds second DNA efficiently. The binding of both gate and transport segments increases with the length of the DNA. Binding of two DNA molecules by the holoenzyme appears to be a cumulative effect of DNA binding to individual subunits. In the absence of any inhibitor, the enzyme accumulates cleaved DNA products with shorter DNA but not with larger DNA. The cleavage of the shorter DNA is supported only in the presence of Mg2+ and Mn2+. Another important property of the enzyme is to introduce positive supercoils which appears to be due to its efficient utilization of ATP and a high rate of reaction. Chapter IV deals with the interaction of mycobacterial gyrase with fluoroquinolones (FQs). Although DNA gyrase is the sole target of the FQs in M. tuberculosis, the lower susceptibility to commonly used FQs have led to the studies to find out more effective quinolones. Previous studies from the laboratory showed a lower susceptibility of the mycobacterial gyrase to ciprofloxacin, but moxifloxacin could inhibit the enzyme efficiently. The better inhibition by moxifloxacin appears to be due to efficient trapping of the enzyme-DNA covalent complex. Both ciprofloxacin and moxifloxacin bind the DNA gyrase from mycobacteria, E. coli and E. coli topo IV, independent of DNA. The extent of binding also correlates with the inhibition potential of the drug against a given enzyme. A general model of quinolone enzyme interaction is provided wherein the quinolones are shown to interact with GyrA subunit or holoenzyme or the enzyme- DNA complex which would finally result in the trapping of the covalent complex.

Packaging of genomic DNA by proteins and super coiling into chromatin and chromatin-like structures (in bacteria) influences nearly all nuclear process such as replication, transcription, repair, and recombination. A ubiquitous class of enzymes termed “DNA topoisomerases” pay key roles during these process. The reactions catalyzed by the members of the DNA topoisomerases family share a common chemistry, which involves phosphodiester bond breakage and re-joining, to bring about a change in the linking number of DNA. Nevertheless, the underlying mechanisms used by these enzymes differ significantly from another. Consequently, DNA topoisomerases are divided into type I and type II enzymes. The mechanism(s) by which DNA topoisomerases perform their functions, and act as targets for anti-bacterial and anti-neoplastic drugs, has attracted considerable interest. Based on these and other finding, I have chosen DNA gyrase from mycobacteria as the subject of my Ph.D. theses investigation. The prokaryotic enzyme, DNA gyrase, is unique amongst all topoisomerases being the only enzyme capable of introducing negative super coils in to duplex DNA. Since no equivalent enzymatic activity has been reported in humans, this essential enzyme has been exploited as a during target against many microbial infections including tuberculosis.DNA gyrase is a tetrameric protein, comprised of two pairs of subunits, encoded by gyrA and gyrB. Inhibitors of DNA gyrase know till date target either of the two subunits and are categorized broadly in to two class, viz. coumarins and quinolones. With the emergence of multiple-drug resistant strains of pathogenic bacteria such as Mycobacterium tuberculosis, which is a leading cause of death world-wide, there is a need to develops new lead molecules with novel mechanisms of inhibition. Towards this end, a new approach to inhibit the mycobacterial DNA gyrase using single-chain antibody has been explore in the present study. In addition to this, the differences in the catalytic properties of the subunits and assembly of the Mycobacterium smegmatis enzyme vis-à-vis Escherichia coli DNA gyrase have been examined. Further, the in vivo relationship of DNA gyrase with the transcription machinery of the cell has also been investigated, with an emphasis on the biology of mycobacteria.

The DNA binding proteins under starvation (Dps) from M. smegmatis are cage-like structures which internalize iron and bind DNA. They provide resistance to the cells from free radical damage, and physically protect the DNA from the harmful effects of reactive oxygen species by DNA compaction. The work compiled in this thesis has been an effort to study oligomerization and dynamics of iron metabolism by these nano-protein compartments. Chapter 1 gives a general introduction on stress, especially oxidative stress, and the ways bacteria fight back the host resistance systems. This has been elaborated from the point of view of the Dps proteins which is the focus of our work. Also, the competition for iron among the host and pathogens, and the modes of iron trafficking of the pathogens from host organisms has been summarized. Finally, the structural aspects of ferritin family proteins to which Dps belongs, has been discussed. Chapter 2 elaborates on the oligomerization pathways of the first M. smegmatis Dps MsDps1, which exists in vitro as two oligomeric forms. The GFP-tagging has been used to locate the Dps1 proteins by live cell imaging and the over-expression of these proteins during nutrient limiting conditions has been studied. The crystal structure of a point mutant F47E in the background of MsDps1, which shows no dodecamerization in vitro, has been solved. The possible ways of dodecamerization of MsDps1 has been concluded by analyzing the intermediates via glutaraldehyde cross-linking and native electrospray mass spectrometry. Chapter 3 documents the gating machinery of iron in MsDps2 protein, the second M. smegmatis Dps protein. Through graph theoretical approaches, a tight histidine-aspartate cluster was identified at the ferritin-like trimeric pore which harbors the channel for the entry and exit of iron. Sitespecific variants of MsDps2 were generated to disrupt this ionic knot, and the mutants were further assayed for ferroxidation, iron uptake and iron release properties. Our studies in MsDps2 show the importance of counter-acting positive and negatively charged residues for efficient assimilation and dispersion of iron. Chapter 4 describes crystallization studies of MsDps2 pore variants, done in an attempt to connect the changes in functional properties described in chapter 3, with structural alterations of the point mutants. We show here that the gating mechanism happens by alterations in side chain configuration at the pore and does not alter the over-all stability of the proteins. Chapter 5 is the final section where we have employed site specific mutations and cocrystallization studies to elucidate the behaviour of MsDps2 proteins upon the addition of iron. By studying the effect of substitutions at conserved sites near ferroxidation center, we attempt to arrive at a pathway which iron atoms take to reach the ferroxidation site. Also, by crystallization of proteins loaded with varying amounts of iron we tried to map the changes in the protein structure in the presence of its ligand. Chapter 6 concludes briefly the work that has been documented in this thesis. Appendix I relates the role of N-terminal tail for DNA binding in MsDp2. Appendix II gives the technical details of a modified protein preparation and oligomerization process for his-tagged MsDps1 protein. Appendix III gives the maps of the plasmids used in this study.