Reengineering the Nucleotide Cofactor Specificity of the RecA Protein by Mutation of Aspartic Acid 100

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 (Roca and Cox, 1990;Kowalczykowski et al., 1994). The presynaptic complex formed between RecA protein and ssDNA is the active recombinational entity in the strand exchange reaction. The RecA protein binds cooperatively to ssDNA, forming a right-handed helical protein filament with one RecA monomer per four nucleotides of ssDNA and six RecA monomers per turn of the filament. In the absence of nucleotide cofactor or in the presence of ADP, the helical filament adopts a "collapsed" or "closed" conformation (helical pitch: 65 Å) that is inactive in strand exchange. In the presence of ATP or the nonhydrolyzable ATP analog, ATP␥S, however, the filament assumes an "extended" or "open" conformation (helical pitch: 95 Å) that is active in strand exchange (Egelman, 1993).We have been examining the mechanism of the nucleotide cofactor-mediated isomerization of the RecA-ssDNA complex and have identified a linkage between the S 0.5 value 2 of a nucleoside triphosphate and the conformational state of the RecA-ssDNA complex (Menge and Bryant, 1992;Meah and Bryant, 1993; Bryant, 1994, 1995). These studies have shown that a nucleoside triphosphate must have an S 0.5 value of 100 -120 M or lower in order to stabilize the strand exchange-active conformation of the RecA-ssDNA complex. For example, although ATP and ITP are hydrolyzed by the RecA protein with identical turnover numbers (18 min Ϫ1 ), ATP (S 0.5 ϭ 45 M) functions as a cofactor for the strand exchange reaction, whereas ITP (S 0.5 ϭ 500 M) is inactive as a strand exchange cofactor.The x-ray crystal structure of the RecA protein-ADP complex indicates that cofactor specificity is determined by Asp 100 , which forms a hydrogen bond with the exocyclic 6-amino group of adenosine base (Story et al., 1992); it seems likely that a similar contact is made with ATP in the RecA-ssDNA-ATP co...

Design and Evaluation of a Tryptophanless RecA Protein with Wild Type Activity

“…Nevertheless, previous studies performed to analyze RecA-cofactor and RecA-DNA interactions have demonstrated the potential power of this approach. For example, Trp replacement of His163 in loop 1 of the RecA protein allowed the formation and conformational dynamics of RecA-ATP␥S filaments to be monitored by real-time spectrofluorometry (15)(16)(17). In addition, mutant proteins such as F203W (12) and Y65W (18) report DNA binding, while Y264W and Y103W (19,20) report binding ATP as well as DNA.…”

A DNA Pairing-enhanced Conformation of Bacterial RecA Proteins

“…Addition of ATP, ATP␥S, or dATP results in a conformation change to an active form that is manifested by extended filaments on DNA with a pitch of 95 Å (41)(42)(43)(44)(45)(46)(47)(48)(49) A, in the 3-strand reaction, a RecA filament is formed on the circular ssDNA. The linear duplex DNA is then aligned with the bound single strand, and exchange is initiated to form a branched intermediate.…”

UTP Is a Cofactor for the DNA Strand Exchange Reaction Performed by the RecA Protein of Escherichia coli

“…Recently, several reports from the Bryant laboratory have shown that binding of a nucleoside triphosphate cofactor to RecA protein bound to ssDNA causes an isomerization of the protein from the inactive to the active form (18,24,36,37). They further showed that the ability of a nucleoside triphosphate cofactor to induce an isomerization in RecA protein to the active form is dependent on the S 0.5 value for that cofactor: the nucleoside triphosphate cofactors whose S 0.5 value is 100-120 µM or lower are capable of stabilizing the strand exchange-active conformation of RecA protein.…”

“…Menetski et al demonstrated that all of the nucleoside triphosphate cofactors that are hydrolyzed by RecA protein are able to induce the high-affinity ssDNA binding state of the RecA, which is the active form of the protein (22). A more recent series of papers from the Bryant laboratory has shown that the ability of a nucleoside triphosphate cofactor to stabilize the active conformation of RecA protein in DNA strand exchange is directly related to the S 0.5 for that cofactor (where S 0.5 is the substrate concentration required for half-maximal velocity) (18,24,36,37). Only the nucleoside triphosphate cofactors whose S 0.5 value is 100-120 µM or lower are capable of stabilizing the strand exchange-active conformation of RecA protein.…”