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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.
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