Mycobacterium smegmatis RecA protein is structurally similar to but functionally distinct from Mycobacterium tuberculosis RecA.
In eubacteria, RecA proteins belong to a large superfamily of evolutionarily conserved, filament-forming, functional homologs of DNA strand exchange proteins. Here, we report the functional characterization of Mycobacterium smegmatis (Ms) and Mycobacterium tuberculosis (Mt) RecA proteins. Although in some respects Ms and Mt RecA proteins are structural and functional homologs of Escherichia coli (Ec) RecA, there are significant differences as well. The single-stranded DNA-binding property of RecA proteins was analyzed by electrophoretic mobility shift assays. We observed that Ms or Mt RecA proteins bound single-stranded DNA in a manner distinct from that of Ec RecA: The former two were able to form protein-DNA complexes in the presence of high salt. Further experiments indicated that Ms or Mt RecA proteins catalyzed adenosine triphosphate hydrolysis at approximately comparable rates across a wide range of pHs. Significantly, DNA strand invasion promoted by Ms or Mt RecA proteins displayed similar kinetics but distinctly different pH profiles. In contrast to MtRecA, MsRecA by itself was unable to form joint molecules across a wide range of pHs. However, regardless of the order in which SSB was added, it was able to stimulate MsRecA to form joint molecules within a narrow pH range, indicating that SSB is a required accessory factor. Together, these results provide a source of sharp contrast between EcRecA and mycobacterial RecAs on the one hand and Mt and Ms RecA proteins on the other.
Ganesh
N
N
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Muniyappa
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism*; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013045:Species Specificity
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases