Differential Rates of NTP Hydrolysis by the Mutant [S69G]RecA Protein

Abstract

The RecA protein of Escherichia coli (M r 37,842, 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. The purified RecA protein will promote a variety of DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing activity is the ATPdependent three-strand exchange reaction in which a circular ssDNA 1 molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule. This reaction proceeds in three phases. In the first phase, the circular ssDNA substrate is coated with RecA protein to form a presynaptic complex; this complex will catalyze the hydrolysis of ATP to ADP and P i . In the second phase, the presynaptic complex interacts with a dsDNA molecule, the homologous sequences are brought into register, and pairing between the circular ssDNA and the complementary strand from the dsDNA is initiated. In the third phase, the complementary linear strand is completely transferred to the circular ssDNA by unidirectional branch migration to yield the nicked circular dsDNA and displaced linear ssDNA products (1).The x-ray crystal structure of the RecA protein indicates that the phosphate groups of the nucleotide cofactor, ATP, are bound by a loop consisting of amino acids 66 through 73 (2). The sequence of this loop ( 66 GPESSGKT 73 ) corresponds to a variation of the well known phosphate binding loop (P-loop) consensus sequence (GXXXXGK[T/S]) found in many NTP-binding proteins (3). The invariant lysine and threonine/serine residues in the P-loop motif are generally found to interact directly with the ␤ and ␥ phosphates of ATP and have been shown for the RecA protein (as well as many other proteins) to be directly involved in the catalysis of phosphoryl transfer (4). Interestingly, although the four variable residues (XXXX) in this sequence can differ widely in different classes of proteins, the specific sequence, GPESSGKT, is highly conserved in over sixty different bacterial RecA proteins (1).As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which the serine residue at position 69 was replaced by a glycine. The biochemical properties of the [S69G]RecA protein provide new insight into the coupling of NTP hydrolysis and DNA strand exchange and are described in this report. EXPERIMENTAL PROCEDURESMaterials-Wild type RecA protein was prepared as described previously (5). rATP was from Sigma; dATP, ddATP, [␣-32 P]rATP, [␣-32 P]dATP, and [␣-32 P]ddATP were from Amersham Pharmacia Biotech. E. coli SSB was from Promega. Circular X ssDNA ((ϩ)-strand) and circular X dsDNA were from New England Biolabs; linear X dsDNA was prepared from circular X dsDNA as described (6). Singleand double-stranded DNA concentrations were determined by absorbance at 260 nm using the conversion factors 36 and 50 g/ml/A 260 , respectively. All DNA concentrations are expressed as total nucleotides....

Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect

“…Although products were clearly in evidence in the wild-type reaction at the 15-min time point, final products did not appear until 60 -90 min after the reaction was initiated with RecA K250R. The slower generation of final products is roughly commensurate with the observed decrease in ATP hydrolysis, and is consistent with the coupling between ATP hydrolysis and DNA strand exchange reported elsewhere (18,58,59). The RecA K250R appears to be fully functional in DNA strand exchange; it is simply slow.…”

“…The mutant protein forms nucleoprotein filaments readily. The fact that the rates of both the ATP hydrolysis and DNA strand exchange are reduced by commensurate amounts reinforces the conclusion of a coupling between ATP hydrolysis and DNA strand exchange that has been evident in previous studies (18,33,58,59,64,65), and it implies a role for Lys-250 in this coupling.…”

Rad51 Uses One Mechanism to Drive DNA Strand Exchange in Both Directions

“…Although the role of ATP hydrolysis in RecA-mediated strand exchange is unclear, it is believed to be important for promoting unidirectional branch migration and to account for its ability to bypass insertions of heterologous sequences (13,(17)(18)(19)24). Furthermore, several groups have shown that there is a direct coupling between NTP hydrolysis and RecA-mediated DNA strand exchange (21,22). However, we have found no indication of coupling between the rate of ATP hydrolysis and branch migration with Rad51.…”

“…ATP Hydrolysis Is Not Correlated with the Rate of Branch-ATP hydrolysis is required for the RecA-promoted strand exchange reaction to proceed in a unidirectional manner and to bypass heterologous insertions (20). Moreover, a correlation between rates of strand exchange and ATP hydrolysis for RecA has been documented (21,22). Rad51's considerably lower ATPase, as compared with that for RecA (6, 7), could account for its lower rate of strand exchange.…”

RecA protein promotes the regression of stalled replication forks in vitro

“…RecA protein is a DNA-dependent ATPase. ATP hydrolysis is clearly coupled to the late phases of RecA protein-mediated DNA strand-exchange reactions in vitro (24,33,41,42). ATP hydrolysis enhances these DNA strand-exchange reactions in several ways, for example allowing the reaction to proceed past structural barriers and incorporate four DNA strands (11,24,36,38,39).…”