Enzymic properties of the RecA803 protein, a partial suppressor of recF mutations

Abstract

The RecA803 protein suppresses the recombinational repair defect of recF mutations and displays enhanced joint molecule formation in vitro (Madiraju et al., 1988). To understand the physical basis for these phenomena, the biochemical properties of RecA803 protein were compared with those of the wild-type protein. The RecA803 protein shows greater DNA-dependent ATPase activity than the wild-type protein with either M13 single-stranded (ss) DNA, which contains secondary structure, or double-stranded DNA. This increased activity reflects an enhanced ability of the mutant protein to form active complexes with these DNA molecules rather than an enhanced catalytic turnover activity, because identical kcat values for ATP hydrolysis are obtained when DNA substrates lacking secondary structure are examined. In addition, the ssDNA-dependent ATPase activity of RecA803 protein displays greater resistance to inhibition by SSB (single-stranded DNA binding) protein. These properties of the RecA803 protein are not due to either an increased binding affinity for ssDNA or an increased kinetic lifetime of RecA803 protein-ssDNA complexes, demonstrating that altered protein-DNA stability is not the basis for the enhanced properties of RecA803 protein. However, the nucleation-limited rate of association with ssDNA is more rapid for the RecA803 protein than for wild-type RecA protein. Consequently, we suggest that altered protein-protein interactions may account for the differences between these two proteins. The implications of these results with regard to the partial suppression of recF mutations by recA803 are discussed (Madiraju et al., 1988).

Reconstitution of initial steps of dsDNA break repair by the RecF pathway of E. coli

“…Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by genesdev.cshlp.org Downloaded from recA mutations (srf, or suppressor of recF) that produce proteins that assemble on ssDNA more rapidly than wildtype RecA (Madiraju et al 1992). One of these suppressor proteins is the RecA730 protein .…”

“…For example, RecO resembles Rad52 with regard to both mediator function and its ability to anneal ssDNA with its cognate SSB/RPA (Kantake et al 2002). Another similarity concerns the RecFOR mediator complex: It functions by loading RecA onto ssDNA complexed with SSB (Morimatsu and Kowalczykowski 2003), and defects in this loading pathway can be suppressed by mutations in RecA (e.g., the RecA803 or RecA730 proteins) that enhance self-assembly on ssDNA (Madiraju and Clark 1990;Madiraju et al 1992;Wang et al 1993). This behavior is paralleled in Saccaromyces cerevisiae (Fortin and Symington 2002), where a hyperactive mutant Rad51 was shown to partially suppress the need for the mediator function of the Rad55/57 complex (Sung 1997), suggesting that RecFOR and Rad55/57 mediator complexes could be functional homologs.…”

A broadening view of recombinational DNA repair in bacteria

“…RecA filaments assemble and disassemble 5 0 to 3 0 in an end-dependent fashion, with a protein being added at one end and deleted at the other (Roca & Cox 1997). Certain mutants of RecA protein suppress the defects of recFOR mutants (Thoms & Wackernagel 1988;Madiraju et al 1992;Wang et al 1993). In vitro work to date indicates that the RecR protein forms alternative complexes with the other two proteins, with different functions (Fig.…”

Biochemical Basis of the Constitutive Coprotease Activity of RecA P67W Protein

“…The enhanced ability to displace SSB protein observed for RecA441, RecA730, and RecA803 proteins was shown to be due to an increased rate of association with ssDNA, as compared to that of wild-type RecA protein (21,22,35,38). The kinetics of RecA protein association were examined using a fluorescence assay which measures the increase in the intrinsic fluorescence of etheno M13 DNA that ac- FIGURE 5: Displacement of SSB protein measured as the appearance of ATP hydrolysis by RecA protein concurs with the direct displacement measurements.…”

“…However, the ATPase activity of RecA441, RecA803, and RecA730 proteins is not inhibited by SSB protein at 1 mM magnesium ion, revealing that these RecA proteins are able to compete with SSB protein for ssDNA binding under these conditions (21,22,38). The ATPase activity of RecA P67W protein was measured at various magnesium ion concentrations to investigate its ability to compete with SSB protein (added after RecA protein addition).…”