Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not.
Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.
Singh
Pawan
P
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka, India.
Tripathi
Pankaj
P
Muniyappa
K
K
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047668:Inteins / Q000502:physiology*; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000201:enzymology* / Q000235:genetics; D055550:Protein Stability; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride
D004269:DNA, Bacterial; D001224:Aspartic Acid; D012965:Sodium Chloride; D011938:Rec A Recombinases; D004720:Endonucleases