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Abstract
The essentiality of DNA Gyrase in basic cellular processes in bacterial pathogens makes it an ideal drug target. Though the Gyrase has a conserved mechanism of action, the complete DNA wrapping and binding process is still unknown. In this study, we have identified six arginine residues R556, R612, R667, R716, R766, and R817 in the DNA GyraseA – C-terminal domain from Salmonella enterica serovar Typhi (StGyrA-CTD) to be essential for DNA wrapping and sliding by a sequence and structure analysis. Through site-directed mutagenesis and EMSA studies, we observed that the substitution of R667 (blade 3) and R716 (blade 4) in StGyrA-CTD led to loss of DNA binding. Whereas, upon mutation of residue R612 (blade2), R766 (blade5) and R817 (blade6) along with supporting residue R712 (blade 4) a decrease in binding affinity was seen. Our results indicate that R667 and R716 act as a pivot point in DNA wrapping and sliding during gyrase catalytic activity. In this study, we propose that the DNA wrapping mechanism commences with DNA binding at blade3 and blade4 followed by other blades to facilitate the DNA sliding during supercoiling activity. This study provides a better understanding of the DNA binding and wrapping mechanism of GyrA-CTD in DNA Gyrase.
Highlights
Topoisomerases are responsible for maintaining the topological state of DNA in the bacterial cell by releasing the torsional stress created during replication, transcription, and recombination[1,2]
The residues involved in these interactions are conserved in the blades of the β-pinwheel structure with the GyrA box determining the geometry of the bound DNA
The arginine residues, R667, R712, R716, R766 and R817 located across the blades are the crucial amino acid residues required for DNA binding (Fig. 5a)
Summary
Topoisomerases are responsible for maintaining the topological state of DNA in the bacterial cell by releasing the torsional stress created during replication, transcription, and recombination[1,2] They are classified as Type I or II based on the number of strands nicked in the initial round of activity. This study based on the sequence alignment of GyrA-CTD from M.tuberculosis with E.coli and B.burgdorferi has identified Y577, D669, R691, and R745 in blade[3] as the residues important for DNA binding in M.tuberculosis(M.tb). We have identified the crucial DNA binding arginine residues involved in Gram negative bacteria using structural and biochemical studies of GyrA-CTD from Salmonella enterica serovar Typhi. Based on the structural and sequence analysis of GyrA-CTD, we have identified 6 conserved arginine residues, one from each blade which plays a significant role in DNA binding and wrapping. R667 and R716 located in blade[3] and blade[4] respectively, as the central pivot point residues for DNA binding and wrapping along with the GyrA box
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