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V. Nagaraja
Ph. D. (IISc., Bangalore, India)
Phone: + 91 80 22932598/ +91 80 23600668
E-mail: vraj@mcbl.iisc.ernet.in

DNA topology modulation, regulation of gene expression and R-M systems are the major areas research with the aim to understand the molecular events and their importance in cellular function

DNA topoisomerases from mycobacteria: Understanding the structure, function and in vivo roles of different topoisomerases in mycobacteria continues to be a major interest in our lab. Both topoisomerase I and DNA gyrase have been studied to unravel their properties which in turn has led to develop strategies to design new class of inhibitors. The highlights include- a. development of peptide inhibitors specific to mycobacterial gyrase with a novel mechanism of action distinct from that of coumarins and quinilones; b. elucidation of properties of gyrase and screeining for new inhibitors; c.unique mode of transcriptional regulation of gyr operon; d. mRNA stability as a means of regulated expression of gyr operon. More recently several gyrase modulatory proteins have been studied.  GyrI and YacG from E. coli were shown to be chromosomally encoded inhibitors of DNA gyrase. YacG is a E. coli gyrase specific inhibitor while GyrI appears to be a repair factor against chromosomal lesions caused by some gyrase poisons. In contrast, glutamate racemase of M. tuberculosis inhibits DNA gyrase from different species. Some of these proteins appear to have evolved specifically for gyrase protection while others such as the racemase might have acquired the moon lighting function.

Our studies have revealed that mycobacterial topoisomerase I is distinct from other prokaryotic and eukaryotic topoisomerases in many characteristics. Unlike other topoisomerases it is site specific and a unique carboxy terminal domain is involved in strand passage reaction. Understanding the need for this specificity and the importance of unusual domains are the current projects.

Biology of R-M systems: We have been addressing the features of site specific interaction, evolution of sequence specificity and emergence of the high specificity enzyme from an ancestral non specific nuclease using R-M systems as a model. Our findings reveal that Kpn1 restriction endonuclease cuts DNA in presence of Ca++, a characteristic not seen with other enzymes. Moreover, Ca++ completely suppresses the unusual high promiscuous activity of the enzyme mediated by Mg++ or Mn++.  An engineered point mutant imparts high specificity cleavage.  This is an example of evolution of sequence specificity by point mutation while in the earlier work we have demonstrated a role for recombination.  Unlike all other Type II enzymes, Kpn1 has H-N-H motif in its active site, found typically in non-specific nucleases.   Thus, Kpn1 appears to be an enzyme evolved from a non specific to sequence specific nuclease. The biological basis for the promiscuous behavior is being addressed.

Regulation of gene expression. Transcription termination: The text book definition of intrinsic transcription terminators is based on E. coli paradigm.  We have demonstrated using both in silico and experimental approaches that intrinsic terminators of various types exist and function in mycobacteria and other bacterial genomes. Unlike the other algorithms, GeSTer developed by us is versatile for finding terminators in genome wide search in all eubacteria. Currently, the algorithm is applied to identify terminators from all sequenced bacterial genomes. We are also investigating the process of Rho factor dependent termination and the possible connection and interplay between the two mechanisms.

Mechanism of transcription activation: We have discovered new features of transcription regulation using mom gene of phage Mu as the system. Hall mark features of the transactivation mechanism elucidated by us are – a. high affinity binding and asymmetric distortion of DNA by transactivator C protein. b. Reorientation of sub-optimal promoter elements for RNAP recruitment. c. intrinsic curvature around –10 which acts as a negative element for RNAP binding. d. The direct interaction of the activator with the transcription machinery is necessary at post recruitment step interactions for enhanced activation.  Activator action in multiple steps is an uncommon feature and seems to have important functional bearing in developmental regulation of phage Mu. The action of transcription activator at promoter clearance step is further investigated by isolating the mutants in polymerase and the transactivator.

In conclusion, our research efforts continue on major DNA transaction processes. The emphasis now has shifted to understand topology modulation by topoisomerase associated proteins.  Orchestrated participation of various factors during different stages of transcription is a subject of major study. Evolution molecular intracellular interactions and their role in specific function is another emerging topic.

Selected Publications

Vos, S.M., Lyubimov, A.M., Hershey,D.M., Schoeffler,A.J., Nagaraja,V  and Berger, J(2014) Control of Topoisomerase-Dependent Supercoiling Homeostasis by an Energy-Linked Feedback System. Genes and Development (InPress)

Bhowmick,T., Ghosh, S., Ramagopal,U.A.,Dey, D.  Ramakumar,S. and Nagaraja,V.  (2014) Targeting Mycobacterium tuberculosis nucleoid associated protein HU by structure based inhibitors. Nature Communications. (In Press)

Manjunatha,U.H., Maxwell, A and  Nagaraja, V. (2005)  A monoclonal antibody that inhibites mycobacterial DNA gyrase by a novel mechanism. Nucl. Acids Res. 33, 3085-3094.

Jain, P. and Nagaraja , V. (2005) An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity. Mol.  Microbiol. 58, 1392-1405.

Jain, P. and Nagaraja , V .(2006) Indispensable, functionally complementing N- and C-  terminal domains   constitute site specific topoisomerase I . J. Mol. Biol. 357: 1409-1421.

Chakraborty, A., and Nagaraja, V. (2006) Dual role for transactivator protein C in activation of mom promoter of bacteriophage Mu.  J. Biol. Chem. 281, 8511-8517.

Sengupta,S., Shah, M. and  Nagaraja V. (2006) Glutamate Racemase from  Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA binding. Nucl. Acids Res. 34, 5567-5576.

Saravanan, M., Vasu, K. and Nagaraja, V. (2008) Evolution of sequence specificity in a restriction endonuclease by a point mutation.Proc. Natl. Acad. Sci, USA. 105, 10344-10347.

List of Publications