Purification and properties of the RecR protein from Bacillus subtilis 168.
Genetic evidence suggests that the Bacillus subtilis recR gene product is involved in DNA repair and recombination. To assign a biochemical function to the recR gene product, the RecR protein was labeled and purified by monitoring the radioactive label. NH2-terminal protein sequence analysis of RecR was consistent with the deduced amino acid sequence of the recR gene. The RecR protein (molecular mass of 25 kDa, isoelectric point 5.4) bound single- and double-stranded DNA in a filter binding assay. RecR-DNA complex formation is enhanced by the presence of a damage in the DNA substrate. The RecR-DNA complex formation proceeds in the absence of divalent cations and nucleotide cofactors, but is markedly stimulated by ATP and divalent cations. In our experimental conditions the apparent equilibrium constants of the optimized RecR-DNA complexes are 3 x 10(-7) M and 9 x 10(-7) M for damaged and undamaged DNA, respectively. The binding reaction is cooperative. Electron microscopy studies show that the presence of divalent cations increases the rate of RecR protein self-assembly. Addition of ATP or dATP promotes the organization of discrete series of quaternary structures on DNA, but ATP gamma S inhibits the DNA binding activity. A possible mechanism for the RecR function in DNA repair is discussed.
Alonso
J C
JC
Max-Planck-Institut für molekulare Genetik, Berlin, Germany.
Stiege
A C
AC
Dobrinski
B
B
Lurz
R
R
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C489318:RecR protein, E coli; D011994:Recombinant Proteins; C065831:RecR protein, Bacteria