Complete Inhibition of Streptococcus pneumoniae RecA Protein-catalyzed ATP Hydrolysis by Single-stranded DNA-binding Protein (SSB Protein)

Abstract

The ATP-dependent three-strand exchange activity of the Streptococcus pneumoniae RecA protein (RecA(Sp)), like that of the Escherichia coli RecA protein (RecA(Ec)), is strongly stimulated by the single-stranded DNA-binding protein (SSB) from either E. coli (SSB(Ec)) or S. pneumoniae (SSB(Sp)). The RecA(Sp) protein differs from the RecA(Ec) protein, however, in that its ssDNA-dependent ATP hydrolysis activity is completely inhibited by SSB(Ec) or SSB(Sp) protein, apparently because these proteins displace RecA(Sp) protein from ssDNA. These results indicate that in contrast to the mechanism that has been established for the RecA(Ec) protein, SSB protein does not stimulate the RecA(Sp) protein-promoted strand exchange reaction by facilitating the formation of a presynaptic complex between the RecA(Sp) protein and the ssDNA substrate. In addition to acting presynaptically, however, it has been proposed that SSB(Ec) protein also stimulates the RecA(Ec) protein strand exchange reaction postsynaptically, by binding to the displaced single strand that is generated when the ssDNA substrate invades the homologous linear dsDNA. In the RecA(Sp) protein-promoted reaction, the stimulatory effect of SSB protein may be due entirely to this postsynaptic mechanism. The competing displacement of RecA(Sp) protein from the ssDNA substrate by SSB protein, however, appears to limit the efficiency of the strand exchange reaction (especially at high SSB protein concentrations or when SSB protein is added to the ssDNA before RecA(Sp) protein) relative to that observed under the same conditions with the RecA(Ec) protein.

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

“…B. subtilis, S. pneumoniae) show a higher activity with dATP as the nucleotide co-factor. RecA⅐dATP⅐Mg 2ϩ (referred to here as RecA⅐dATP) or RecA Spn ⅐dATP catalyzes DNA strand exchange even in the absence of accessory factors (11)(12)(13)(14). The RecA⅐dATP transitional states resemble those described previously for RecA Eco , designated as O, Ac, Ao, and P states ( Fig.…”

“…Similarly, other RecA proteins from naturally transforming bacteria (e.g. RecA Spn or RecA Dra ) in the ATP-bound form are unable to catalyze DNA strand exchange (12,13,33).…”

“…RecA, RecA Spn , or RecA Dra ) cannot catalyze DNA strand exchange in the absence of accessory proteins (11)(12)(13)(14)33). A DNA strand exchange assay that uses circular ssDNA and linear duplex DNA was employed to investigate whether SsbA or SsbB helps overcome the inability of RecA⅐ATP to catalyze DNA recombination.…”

“…19 and 20 and our unpublished results), but the relative affinity of RecA for ATP or dATP is similar (21), suggesting that the catalytic site of RecA would contain ATP primarily. This premise creates a paradox: RecA⅐ATP can nucleate onto ssDNA, but it cannot catalyze DNA strand exchange (11,12,14). Furthermore, RecA⅐ATP neither nucleates nor polymerizes onto SsbA-coated ssDNA (14).…”

Differential Single-stranded DNA Binding Properties of the Paralogous SsbA and SsbB Proteins from Streptococcus pneumoniae

“…The shorter C-terminal region of the SsbB protein, on the other hand, may not only modify the ssDNA binding properties, but may also alter the spectrum of protein-protein interactions that are available to the SsbB protein and enable it to function more effectively in conjunction with various recombination proteins during natural transformation. The ssDNA binding studies that are presented in this report, together with the recent isolation and characterization of other proteins that have been implicated in transformational recombination (12,13), will provide a foundation for further investigations into the biological roles of the paralogous SsbA and SsbB proteins.…”