Characterization of Single-stranded DNA-binding Proteins from Mycobacteria

Abstract

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

RecX protein abrogates ATP hydrolysis and strand exchange promoted by RecA: Insights into negative regulation of homologous recombination

“…Polyclonal antibodies against EcRecA and M. tuberculosis RecX were generated in rabbits. Anti-EcRecA antibodies cross-reacted with MtRecA (4), as well as MsRecA (14). Anti-RecX antibodies cross-reacted with purified RecX, as well as a 19-kDa protein in the cell lysates of M. tuberculosis and M. smegmatis.…”

“…Under these conditions, 10 ng of [ 3 H]ssDNA incubated with the lysates was completely degraded to acid-soluble nucleotide residues by 10 g of DNaseI (data not shown). Immunoprecipitation and immunoblotting experiments were performed as described (14). An aliquot of cell lysate (0.5-0.75 mg protein) in 20 mM Tris⅐HCl (pH 7.5)͞0.2 M NaCl͞1 mM ATP (buffer TNA) was incubated with anti-RecA or anti-RecX antibodies at 4°C for 6 h, and then with 50 l of slurry of protein A-Sepharose for an additional 2 h. Immunoprecipitates were collected by centrifugation, washed six times with TNA buffer containing 0.1% Triton X-100, stained with appropriate primary and secondary antibody, and visualized by chemiluminescence (14,20).…”

“…The negative regulation of RecA by RecX implies that RecX might act as an antirecombinase to quell inappropriate recombinational repair during normal DNA metabolism. (14) and their concentrations determined as described (15). Negatively supercoiled (form I) and circular single-stranded M13 DNA (ssDNA) was prepared as described (16).…”

Effect of Single-stranded DNA-binding Proteins on the Helicase and Primase Activities of the Bacteriophage T7 Gene 4 Protein

“…Strand transfer mediated by the gene 4 helicase readily occurs in the presence of gp2.5 (14). T7 gp2.5, E. coli SSB protein, and T4 gene 32 protein all have an acidic C terminus of which a number of studies shown are essential for the protein-protein interactions observed in vitro (6,7,13,(15)(16)(17)(18)(19). A truncated gp2.5, gp2.5-⌬21C, lacking the 21 C-terminal residues cannot support T7 growth, and the purified protein cannot form dimers or physically interact with T7 DNA polymerase or gp4 (6).…”

Variations in amino acid composition in bacterial single stranded DNA–binding proteins correlate with GC content

“…These strands form two additional clamp-like structures in the homotetrameric SSB which contribute to the overall stability of the quaternary structure of SSB (18,21). It is thought that the mode of DNA binding of actinobacterial SSBs is different from that of EcoSSB partly on account of the difference in the shape of the tetramers (22,23).…”