Effect of nucleotide cofactor structure on RecA protein-promoted DNA pairing. 1. Three-strand exchange reaction

Abstract

The structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), are all hydrolyzed by the recA protein with the same turnover number (17.5 min-1). The S0.5 values for these nucleotides increase progressively in the order ATP (45 microM), PTP (100 microM), ITP (300 microM), and GTP (750 microM). PTP, ITP, and GTP are each competitive inhibitors of recA protein-catalyzed ssDNA-dependent ATP hydrolysis, indicating that these nucleotides all compete for the same catalytic site on the recA protein. Despite these similarities, ATP and PTP function as cofactors for the recA protein-promoted three-strand exchange reaction, whereas ITP and GTP are inactive as cofactors. The strand exchange activity of the various nucleotides correlates directly with their ability to support the isomerization of the recA protein to a strand exchange-active conformational state. The mechanistic deficiency of ITP and GTP appears to arise as a consequence of the hydrolysis of these nucleotides to the corresponding nucleoside diphosphates, IDP and GDP. We speculate the nucleoside triphosphates with S0.5 values greater than 100 microM will be intrinsically unable to sustain the strand exchange-active conformational state of the recA protein during ongoing NTP hydrolysis and will therefore be inactive as cofactors for the strand exchange reaction.

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

“…4, the wild-type RecA protein has full strand exchange activity in the presence of ATP (3 mM), but has no detectable activity in the presence of ITP (3 mM), consistent with our previous results (Menge and Bryant, 1992). In contrast, the [D100N]RecA protein exhibited full strand exchange activity in the presence of either ATP or ITP (3 mM).…”

“…Materials-Wild-type RecA protein was prepared as described previously (Cotterill et al, 1982 P]ATP and nucleoside diphosphate kinase (Sigma) as described previously (Menge and Bryant, 1992). E. coli SSB was from Pharmacia Biotech Inc.. 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 (Cox and Lehman, 1981).…”

“…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.…”

“…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.…”

Biochemical and Cellular Characterization of Helicobacter pylori RecA, a Protein with High-Level Constitutive Expression

“…Despite the similarities between HpRecA and EcRecA, HpRecA seems to be less efficient in vitro than its homolog, since its kinetic constants are reduced 2-to 4-fold (Fig. 1C) (19). Nevertheless, the constants are in the same range as those of Bacillus subtilis RecA (BsRecA) (29).…”