DNA topoisomerases and topology modulation
Topoisomerases together with the nucleoid associated proteins (NAPs) ensure bacterial chromosome compaction to regulate all major DNA transaction processes. While we continue our studies on function and in vivo roles of both DNA gyrase and topoisomerase I (TopoI) from mycobacteria, understanding the function of different NAPs is now a major topic. Highlights of the work over the last 10 years are: Development of peptide inhibitors specific to mycobacterial gyrase with a novel mechanism of action, elucidation of dual role of gyrase in supercoiling and decatenation, the molecular basis of switch from supercoiling to decatenation function- binding of second DNA to GyrB subunit, demonstration of direct binding of fluoroquinolones to GyrA subunit and holoenzyme in addition to the established mode of action, action of gyrase modulatory protein, YacG, in regulating the supercoiling activity by inhibiting strand passage, importance of carboxyl terminal basic residues in strand passage activity of TopoI, development of inhibitory monoclonal antibodies against TopoI and first set of small molecule inhibitors, characterization of a membrane anchored NAP (Rv3852) involved in lipid metabolism, biofilm formation, M. tuberculosis (Mtb) HU structure based inhibitor design, chemical perturbation of Mtb and inhibition of cell growth. A direct physical interaction between HU and TopoI to enhance DNA relaxation activity of the enzyme by stimulating the strand passage step is demonstrated. Given the underrepresentation of topoisomerases and NAPs in Mtb, such functional collaborations may be necessary to maintain the topological homeostasis. New NAPs are being characterized. Further, we have begun to understand the importance of posttranslational modification of these topology modulators. To start with, an acetyl transferase which extensively acetylates HU is identified and other protein modifying enzymes are being characterized.
Regulation of gene expression in mycobacteria
To study transcription initiation, elongation and termination in mycobacteria, we developed a method for purification of optimized RNAP holoenzyme (with stoichiometric sigma factor). Promoter:Polymerase interaction studies with this enzyme revealed different rate limiting steps at various promoters. Surprisingly, gyr promoters for the same genes from M. smegmatis and Mtb showed contrastingly different properties. We have also elucidated the distinct mechanism of growth phase dependent regulation of gyr and rRNA promoters of Mtb. An interaction of different set of residues in the promoter discriminator region and sigma 1.2 region dictate the initiating NTP and pppGpp mediated regulation of transcription initiation at these promoters. The mechanism of autoregulation of gyrase and topoisomerase I have been elucidated. The connection between relaxation stimulated transcription and reiterative transcription is being established. We have also investigated both intrinsic and factor dependent transcription termination, uncovering alternate paradigms and unusual characteristics. Rho is abundant in mycobacteria and understanding new facets of Rho function has led us to suggest it as the major regulator in the genus. Currently, we have begun to explore co-operation between intrinsic and Rho mediated termination. As a follow up, we have generated conditional knock down strains of key cellular players described above to investigate the topology - transcription connection.
Regulation of mom and understanding its function
Earlier, our efforts were to understand the complex regulation of Mom, the unique anti-restriction function of Phage Mu. After elucidating the mode of multistep transcription activation (an example of irreversible genetic switch) of the otherwise silenced gene, we have discovered FIS (a host encoded NAP) mediated transcription silencing of mom. In parallel, a multipronged approach is undertaken to understand the biochemistry of Mom function. Efforts are underway to find the co-factor of Mom and the nature of enzyme activity responsible for the unique DNA modification.
Biology of R-M systems:
We have pursued our interests on site specific interaction, evolution of sequence specificity and engineering specific enzyme from an ancestral nonspecific nuclease using R-M systems as a model. Although restriction enzymes are believed to be exquisitely sequence specific, our studies with KpnI revealed high degree of promiscuity. The biological basis for the promiscuous behaviour was addressed and we demonstrate that the promiscuity of the enzyme provides an advantage to the host against the invading phages, a winning strategy in the co-evolutionary arms race between them. Given the presence of a large number of R-M systems in diverse bacteria and the fact that R-M mediated innate defense is inadequate in the arms race with the invading genomes, we have considered other roles for this diverse group of nucleases. These additional cellular roles could be in controlling the phage proliferation (after the phages have overcome restriction barrier) and in bacterial apoptosis – an altruistic social behaviour benefiting the community at large. These models are being tested.