Exploration of the Recombination Repair Pathway in Mycobacteria : Identification and Characterization of New Proteins

Abstract

Genomic integrity is a fundamental requisite for survival and proliferation of all organisms. The genetic material is continuously threatened by a multitude of extrinsic and intrinsic factors. Consequently, the presence of strong DNA repair systems is essential to aid errorfree transmission of genetic material to successive generations. In prokaryotes, repair by homologous recombination (HR) provides a major means to reinstate the genetic information lost in DNA damage. Pathogenic bacteria, such as Mycobacterium tuberculosis, face an additional threat of DNA damage due to antibiotic treatment and immune stresses inside the macrophage. Consequently, M. tuberculosis has evolved a remarkably strong DNA repair network, providing it robust survivability in the harsh environments faced inside the host cell. The importance of HR or recombination repair in pathogenesis and emergence of antibiotic resistance in M. tuberculosis is well established. However, many aspects of the pathway remain elusive as of now. This thesis is concerned with the analysis of recombination repair system in the genus mycobacteria and characterization of two novel proteins identified in the process. Chapter 1 gives a detailed account of the mechanics of HR, using the well studied E. coli system and highlights the differences in mycobacteria. Recombination repair comprises a series of processes carried out by more than 20 proteins, that ultimately leads to repair of damaged DNA. Processing of DNA strands at double strand breaks and single strand gaps, to produce a 3' overhang, initiates the process. Unlike E. coli, complexes of RecFO-RecR and AdnAB-RecR provide two alternate pathways for end resection of strands and RecA loading in mycobacteria. The exchange of an undamaged strand with the damaged strand, facilitated by RecA, is central to recombination. Additionally, Single-Stranded DNA binding proteins (SSB) facilitate the loading of RecA onto the single-stranded overhang produced by the pre-processing enzymes. Resolution of strands formed due to strand exchange, via multi-stand branched DNA intermediates (such as D-loops, three-way junctions, Holliday junctions etc) by RuvABC or RecG, is the final step of recombination. An additional HJ resolvase YqgF, with unclear functions, is also present in mycobacteria. Furthermore, the major end resection enzymes (RecBCD) involved in HR in E. coli, were implicated in the Single-Strand Annealing (SSA) pathway in mycobacteria. As part of an effort to improve the understanding of recombination repair in mycobacteria, a structure based genomic search for such proteins was carried out in 43 mycobacteria with known genome sequences (Chapter 2). Of about 20 proteins known to be involved in the pathway, a set of 9 proteins, namely, RecF, RecO, RecR, RecA, SSBa, RuvA, RuvB and RuvC was found to be indispensable among the 43 mycobacterial strains. A domain level analysis indicated that most domains involved in recombination repair are unique to these proteins and are present as single copies in the genomes. Synteny analysis reveals that the gene order of proteins involved in the pathway is not conserved, suggesting that they may be regulated differently in different species. Sequence conservation among the same protein from different strains suggests the importance of RecO-RecA and RecFORRecA presynaptic pathways in the repair of double strand-breaks and single strand-breaks respectively. New insights into the binding of small molecules to the relevant proteins are provided by binding pocket analysis using three-dimensional structural models. New annotations obtained from the analysis, include identification of a protein (RecGwed) with a probable Holliday junction binding role present in 41 mycobacterial genomes and that of a RecB-like nuclease, containing a cas4 domain, present in 42 genomes. A second SingleStranded DNA Binding protein (SSBb), in addition to the canonical one (SSBa), was present in all mycobacteria except M. leprae. Chapter 3 describes the cloning, expression, purification and structural studies on SSBb from M. smegmatis (MsSSBb). MsSSBb has been crystallized and X-ray analyzed in the first structure elucidation of a mycobacterial SSBb. The protein crystallizes in hexagonal space group P6522 (a = b =73.61 Å, c = 216.21 Å), with half a tetrameric molecule in the asymmetric unit of the cell. In spite of the low sequence identity between SSBas and SSBbs in mycobacteria, the tertiary and quaternary structure of the DNA binding domain of MsSSBb is similar to that observed in mycobacterial SSBas. In particular, the quaternary structure is 'clamped' using a C-terminal stretch of the N-domain, which endows the tetrameric molecule with additional stability and its characteristic shape. A comparison involving available, rather limited, structural data on SSBbs from other sources, appears to suggest that SSBbs could exhibit higher structural variability than SSBas do. It was realized that many bacterial species have a paralogous SSBb. The SSBb proteins have not been well characterized. While in B. subtilis, SSBb has been shown to be involved in genetic recombination; in S. coelicolor it mediates chromosomal segregation during sporulation. Chapter 4 describes the distinct properties and the role of SSBb in mycobacteria. Sequence analysis of SSBs from mycobacterial species suggests low conservation of SSBb proteins, as compared to the conservation of SSBa proteins. Like most bacterial SSB proteins, M. smegmatis SSBb (MsSSBb) forms a stable tetramer. However, solution studies indicate that MsSSBb is less stable towards thermal and chemical denaturation than MsSSBa. Also, in contrast to the 5-20 fold differences in DNA binding affinity between paralogous SSBs observed in other organisms, MsSSBb is only about twofold poorer in its DNA binding affinity than MsSSBa. The expression levels of ssbB gene increased during UV and hypoxic stresses, while the levels of ssbA expression declined. A direct physical interaction of MsSSBb and RecA, mediated by the C-terminal tail of MsSSBb was also established. The results obtained in this study indicate a role of MsSSBb in recombination repair during stress. Chapter 5 describes the characterization of the previously annotated hypothetical protein RecGwed and its probable role as a novel regulator in the resolution of branched DNA structures. The protein is composed of an unusually charged N-terminus and a C-terminal 'wedge' domain, similar to the wedge domain of RecG. A database search suggested that RecGwed is predominately present in the phylum Actinobacteria, along with some other known human pathogens. Purified M. smegmatis RecGwed (MsRecGwed) exists as a stable monomer in the solution. CD studies and homology modeling indicated an unusually low content of regular secondary structures. MsRecGwed was able to bind branched DNA structures such as Holliday junction, three-way junction, three-strand junction and replication fork in vitro, while it does not interact with ss- or dsDNA. The expression of recGwed in M. smegmatis was up-regulated during stationary phase/UV damage and down-regulated during MMS/H2O2 treatment. These observations indicate the possibility of involvement of RecGwed in DNA transactions in post-replicative (stationary phase) recombination events, that proceed though branched DNA intermediates. The work described in this chapter is the first report of characterization of RecGwed-like proteins. Taken together, the work done in this thesis augments the existing repertoire of proteins known to be involved in DNA repair pathways in mycobacteria. As indicated in the concluding chapter, this study also creates a trail of future experiments that will improve our current understanding of HR in mycobacteria. As a part of ongoing efforts in the laboratory, on the characterization of enzymes which sanitize the nucleotide pool to prevent DNA damage, structural studies on M. smegmatis MutT2 have been carried out (Appendix). Structure of the native protein, and its complexes with substrates 5me-dCTP, dCTP and CTP and the respective products, has been determined. The work presented here is the first report of MutT2-type CTP pyrophosphorylase enzymes in complex with substrates. It provides insights into the mechanism of action and the molecular basis of the functioning of mycobacterial MutT2