Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn(2+) and DNA-dependent ATPase activity.
Mycobacterium tuberculosis RecA intein (PI-MtuI), a LAGLIDADG homing endonuclease, displays dual target specificity in response to alternative cofactors. While both ATP and Mn(2+) were required for optimal cleavage of an inteinless recA allele (hereafter referred to as cognate DNA), Mg(2+) alone was sufficient for cleavage of ectopic DNA sites. In this study, we have explored the ability of PI-MtuI to catalyze ATP hydrolysis in the presence of alternative metal ion cofactors and DNA substrates. Our results indicate that PI-MtuI displays maximum ATPase activity in the presence of cognate but not ectopic DNA. Kinetic analysis revealed that Mn(2+) was able to stimulate PI-MtuI catalyzed ATP hydrolysis, whereas Mg(2+) failed to do so. Using UV crosslinking, limited proteolysis and amino acid sequence analysis, we show that (32)P-labeled ATP was bound to a 14 kDa peptide containing the putative Walker A motif. Furthermore, the limited proteolysis approach disclosed that cognate DNA was able to induce structural changes in PI-MtuI. Mutation of the presumptive metal ion-binding ligands (Asp122 and Asp222) in the LAGLIDADG motifs of PI-MtuI impaired its affinity for ATP, thus resulting in a reduction in or loss of its endonuclease activity. Together, these results suggest that PI-MtuI is a (cognate) DNA- and Mn(2+)-dependent ATPase, unique from the LAGLIDADG family of homing endonucleases, and implies a possible role for ATP hydrolysis in the recognition and/or cleavage of homing site DNA sequence.
Guhan
N
N
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Muniyappa
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding / Q000187:drug effects; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*
D008345:Manganese; D000255:Adenosine Triphosphate; D004247:DNA; D011938:Rec A Recombinases; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D008274:Magnesium