Abstract
Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.
Highlights
Complexesformedfrom A13+or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein
The RecA protein of Escherichia coli has been shown to play an essential role in genetic recombination and in the induction of at least 17 genes whichare negatively controlled by the LexA protein (Walker,1985).The latteractivity of the RecA protein results from its capacity to promote cleavage of the LexA repressor (Slilaty and Little, 1987).RecA-promoted proteolysis is highly specific because, besides the LexA protein, the RecA protein promotes the cleavage of one other bacterial protein, UmuD (Burckhardt et al, 1988), and that of bacteriophage repressors, such as theX c l repressor (Roberts and Roberts, 1981); all these proteins share structural homologies (Nohmi et al, 1988)
In order to investigate these processes, we studied the effects of structural analogues of phosphate, the fluoride complexes of aluminum and beryllium, on two RecA-dependent reactions, namely ATP hydrolysis and cleavage of LexA protein
Summary
Complexesformedfrom A13+or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. It has been proposed that AlF; and BeF; complex ions, which have the same tetrahedral geometry and bond length as inorganic phosphate, act by mimickingthe y-phosphate of ATP or GTP, thereby converting various enzymes from a conformation normally observed with GDP or ADP to a conformation observed with nucleoside triphosphates (Sternweis and Gilman, 1982; Bigayet al., 1985; Lange et al, 1986; Robinson et al, 1986; Bigay et al, 1987; Carlier et al, 1988) These analogues may be useful to probe the catalytic mechanism of nucleotidases involved in energy transduction
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