ADP-dependent DNA Strand Exchange by the Mutant [P67G/E68A]RecA Protein

Abstract

We have prepared a mutant RecA protein in which proline 67 and glutamic acid 68 in the NTP binding site were replaced by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of ATP and is able to promote the standard ATP-dependent threestrand exchange reaction between a circular bacteriophage X174 (X) single-stranded DNA molecule and a homologous linear X double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the wild type RecA protein, however, in that it is (i) completely inhibited by an ATP regeneration system, and (ii) strongly stimulated by the addition of high concentrations of ADP to the reaction solution. These results indicate that the strand exchange activity of the [P67G/E68A]RecA protein is dependent on the presence of both ATP and ADP. The ADP dependence of the reaction is reduced or eliminated when (i) a shorter linear X dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear X dsDNA substrate, or (ii) the Mg 2؉ concentration is reduced to a level just sufficient to complex the ATP present in the reaction solution. These results indicate that it is the branch migration phase (and not the initial pairing step) of the [P67G/E68A]RecA protein-promoted strand exchange reaction that is dependent on ADP. It is likely that the [P67G/E68A]RecA mutation has revealed a requirement for ADP that also exists (but is not as readily apparent) in the strand exchange reaction of the wild type RecA protein.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 promotes a variety of ATP-dependent DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing reaction is the 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 molecule is coated with RecA protein, forming a helical nucleoprotein filament known as the presynaptic complex. In the second phase, the presynaptic complex interacts with the linear 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, 2).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 -73 (3). The sequence of this loop ( 66 GPESSGKT 73 ) corresponds to a variation of the well known phosphate binding loop...