| dc.description.abstract | Mycobacterium smegmatis TOPOISOMERASE I: INSIGHTS INTO SITE-SPECIFIC RECOGNITION AND MECHANISM OF ACTION
The work embodied in this thesis is an attempt to understand the relaxation mechanism catalyzed by Mycobacterium smegmatis topoisomerase I. For this purpose, a detailed study was undertaken that involved identification of preferred binding sites in the mycobacterial genome and deciphering the molecular basis of preferential interaction at these sites. The site specificity of the enzyme allowed designing defined substrates for addressing the basis of Mg(II) requirement during the catalytic cycle of relaxation. In addition, efforts were directed towards the identification of proteins that could modulate topoisomerase I activity. The salient aspects are summarized below.
As an introduction, Chapter I is a review of the literature that describes the identified topoisomerases, their classification, properties, and the present understanding of their multifarious roles, which has led to considering them as global regulators of crucial macromolecular events. The reaction mechanism of various classes of topoisomerases is briefly discussed. This chapter also deals with a comparative analysis of structural aspects of various topoisomerases, reflecting the commonality between type IA and type II topoisomerases. Major developments in the field with respect to novel activities of topoisomerases and their interaction with other cellular proteins have also been discussed.
Chapter 2 deals with the detailed study carried out in order to identify Strong Topoisomerase Sites (STS) from the genomic sequences of Mycobacterium tuberculosis and M. smegmatis. A novel strategy was designed for this purpose. The study reveals that the enzyme does not nick DNA in a random fashion; DNA cleavage occurs at a few specific sites. Mapping of these sites revealed conservation of a hexanucleotide sequence CG/TCTTC/G at the cleavage site. The enzyme binds and cleaves consensus oligonucleotides having this sequence motif. An important finding is that the protein exhibits a very high preference for a C or G residue at the +2 position with respect to the cleavage site. It appears that the enzyme functions in vivo mainly at these specific sites to carry out topological reactions.
In Chapter 3, sequence-specific interaction of Mycobacterium smegmatis topoisomerase I at its recognition sequence is elucidated. High-resolution footprinting analysis using a variety of probes allowed defining the determinants of this interaction. DNase I footprinting demonstrates a large region of protection on both the scissile and non-scissile strands of DNA. Methylation protection and interference analyses reveal base-specific contacts. Missing contact analyses suggest a similar interaction with the residues in single- and double-stranded DNA and emphasize the role of functional groups associated with those bases, rather than groove specificity. These interactions are supplemented by essential phosphate contacts in the scissile strand. Conformation-specific probes reveal protein-induced structural distortion of the DNA helix at the ‘T-A-T-A’ sequence 11 bp upstream of STS. Based on this high-resolution footprinting analysis, which defines topoisomerase-DNA interactions, a model of topoisomerase I binding to its substrate is presented. The protein binds a stretch of 30 bp within which lie two regions of DNA that provide major contacts. While the first is localized around the STS, the second lies 8 bases upstream. The enzyme thus makes asymmetric recognition on DNA and seems to carry out DNA relaxation either by enzyme-bridged or restricted rotation mechanism.
Mg(II) ions are important for DNA transaction enzymes as they either alter the structure of proteins or influence the catalytic step of the reaction. While the divalent cation is essential for type IA topoisomerase activity, it only stimulates type IB enzymes. In Chapter 4, the requirement of Mg(II) during the relaxation reaction catalyzed by Mycobacterium smegmatis topoisomerase I is shown. The role of Mg(II) during individual steps of the topoisomerization cycle was addressed. The protein forms non-covalent and covalent complexes but fails to catalyze religation in the absence of the divalent ion. Using defined oligonucleotide substrates, it is demonstrated that the reformation of the phosphodiester bond at the site of cleavage is Mg(II)-dependent. DMS footprints of the topoisomerase-DNA complex reveal a change in the interaction pattern of topoisomerase at its recognition site in the presence of magnesium. Alteration in the protease sensitivity pattern indicates a change in protein conformation in the presence of Mg(II) ions.
Protein-protein interaction plays an important role by favorably or adversely influencing major molecular events. In most documented cases, the interaction is direct between the partner molecules. Influence on activity in the absence of direct physical interaction between DNA transaction proteins is another important means of modulation. Chapter 5 deals with stimulation of topoisomerase I activity by single-stranded binding protein (SSB) without direct protein-protein interactions. The stimulation is specific to topoisomerase I, as DNA gyrase activity is unaffected by SSB. Such cases of functional collaboration between DNA transaction proteins could be of common occurrence in important molecular processes. The above example is unlikely to be an exception. | |
