| dc.description.abstract | RecA protein plays a crucial role in homologous genetic recombination, DNA repair, and SOS induction. Homologous recombination involves genetic exchange between any two DNA molecules sharing extended regions with homologous sequences. Maintaining the integrity of information in DNA is essential for survival. It requires the existence of an elaborate repair system in the cell. Extensive DNA damage triggers a response in the cell involving approximately 20 genes. This process is known as the SOS response. RecA is a moderately sized protein with a molecular weight of 38 kDa, involved in recombination, repair, and SOS response. It forms helical filaments both in the presence and absence of DNA. It binds to both single-stranded and double-stranded DNA, and binds and hydrolyses ATP in a DNA-dependent manner. It facilitates the auto-cleavage of the LexA repressor, which plays a crucial role in the SOS response. It promotes the strand exchange reaction.
The work reported in this thesis is concerned with structural studies on RecA from two mycobacteria, namely Mycobacterium tuberculosis and Mycobacterium smegmatis, and their nucleotide complexes.
The hanging-drop vapour diffusion technique was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator. The data were processed using the DENZO and SCALEPACK program suites. AMoRe was employed for structure solution using molecular replacement. Structure refinements were carried out using X-PLOR and CNS software packages. Model building was done using the packages FRODO and O. PROCHECK, MSP, ALIGN, and INSIGHT were used for structure validation and analysis of the refined structures.
The crystal structure of M. tuberculosis RecA (MtRecA), the first structure to be determined in the programme, has six molecules in the unit cell forming a 6 helical filament, with a deep groove capable of binding DNA. The observed weakening in the higher-order aggregation of filaments into bundles may have implications for recombination in mycobacteria. The structure of the complex reveals the atomic interactions of ADP–AlF, an ATP analogue, with the P-loop-containing binding pocket. The structures explain reduced levels of interaction of MtRecA with ATP in terms of the expansion of the binding site, despite sharing the same fold, topology, and high sequence similarity with RecA from Escherichia coli (EcRecA). The formation of a helical filament with a deep groove appears to be an inherent property of MtRecA.
The crystal structures of an MtRecA–ADP complex, complexes with ATPS in the presence and absence of magnesium, as well as a complex with dATP and Mg², were subsequently determined. Comparison with the crystal structures of the apo form as well as the complex with ADP–AlF confirms an expansion of the P-loop region in MtRecA compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand-bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve to maintain the network of interactions crucial for nucleotide binding. The nucleotide-binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPS, rather than ADP–AlF, is structurally a better mimic of ATP.
Among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide-binding pocket at the molecular level. Comparison with other NTP-binding proteins reveals many commonalities in modes of binding among diverse members of the structural family, contributing to our understanding of the structural signature of NTP recognition.
The crystal structures of Mycobacterium smegmatis RecA (MsRecA) and its complexes with ADP, ATPS, and dATP were then determined. Comparison of these structures with those of MtRecA reveals the relatively rigid and flexible regions of the mycobacterial RecA molecule. The regions involved in the stability of the structure are relatively rigid, while those involved in function are substantially flexible. The interface involved in the formation of the filament is nearly the same in all available RecA structures, including EcRecA. Bundle formation, however, shows considerable variability, consistent with biochemical results.
The nucleotide-binding regions in the mycobacterial proteins expand with respect to that in EcRecA, providing a structural basis for the reduced affinity of MsRecA and MtRecA for nucleotide cofactors. However, the difference between modes of binding to ATPS and dATP is not the same in MtRecA and MsRecA. The most conspicuous and consistent effect of nucleotide binding is the movement of Gln196, which is the first residue in one of the two DNA-binding loops. This movement appears to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding regions. The conformation and disposition of the DNA-binding loops are different in MsRecA and MtRecA, providing a probable structural basis for the functional diversity in their DNA-binding and strand-exchange properties.
A study involving lysozyme to explore the effect of stabilizing additives on the structure and hydration of proteins, carried out by the author during the period of his studentship, is described in an Appendix. | |
