Abstract
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.
Highlights
Genetic analysis shows that the RecBCD or @ pathway is dependent otnhe RecAR,ecBCD (exonuclease V, termed AddAB(C?) in B. subtilis) and single-stranded DNA-binding proteins (SSB)' [1,2,3,4,5]
What is known about the RecF or cy pathway of homologous recombination? In bothbacteria, mutations in the recF, recR, or recL genes were shown either to inhibit or reduce a variety of DNA metabolic events, e.g. SOS induction, DNA recombination, and postreplication repair [1, 3, 11,12,13,14,15,16]
We show that the binding of the RecR protein to dsDNA is enhanced when the DNA substrate is pretreated with different DNA damaging agents
Summary
Purification ofRecR Protein-The pBT56-encoded RecR protein [17] was specificallylabeled with [36S]methioninewith the help of an in vivo expression system [23]. The RecR polypeptide, under the expression conditions described under "Experimental Procedures," accounts for about 12% of total protein mass. The overproduced 25-kDa polypeptide (predicted molecular weight = 24,298) was insoluble (Fig. L4, lune 2). The RecR aggregates could, be dissolved in thepresence of 2 M urea. This property was exploited in our purification scheme to release unwanted proteins. After the lastpurification step the (25-kDa) RecR polypeptide is more than 99% pure,as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1B, lune 7). Reaction requirementsfor RecR-DNA bindingactivity is increased about 2-fold when ATPor ADP, at afinal
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