Functional characterization of the precursor and spliced forms of RecA protein of Mycobacterium tuberculosis.
The recA locus of pathogenic mycobacteria differs from that of nonpathogenic species because it contains large intervening sequences nested in the RecA homology region that are excised by an unusual protein-splicing reaction. In vivo assays indicated that Mycobacterium tuberculosis recA partially complemented Escherichia coli recA mutants for recombination and mutagenesis. Further, splicing of the 85 kDa precursor to 38 kDa MtRecA protein was necessary for the display of its activity, in vivo. To gain insights into the molecular basis for partial and lack of complementation by MtRecA and 85 kDa proteins, respectively, we purified both of them to homogeneity. MtRecA protein, but not the 85 kDa form, bound stoichiometrically to single-stranded DNA in the presence of ATP. MtRecA protein was cross-linked to 8-azidoadenosine 5'-triphosphate with reduced efficiency, and kinetic analysis of ATPase activity suggested that it is due to decreased affinity for ATP. In contrast, the 85 kDa form was unable to bind ATP, in the presence or absence of ssDNA and, consequently, was entirely devoid of ATPase activity. Molecular modeling studies suggested that the decreased affinity of MtRecA protein for ATP and the reduced efficiency of its hydrolysis might be due to the widening of the cleft which alters the hydrogen bonds and the contact area between the enzyme and the substrate and changes in the disposition of the amino acid residues around the magnesium ion and the gamma-phosphate. The formation of joint molecules promoted by MtRecA protein was stimulated by SSB when the former was added first. The probability of an association between the lack and partial levels of biological activity of RecA protein(s) to that of illegitimate recombination in pathogenic mycobacteria is considered.
Kumar
R A
RA
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Vaze
M B
MB
Chandra
N R
NR
Vijayan
M
M
Muniyappa
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014774:Virulence
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D011498:Protein Precursors; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases