Biochemical Characterization of the Human RAD51 Protein

Abstract

Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially (ϳ10-fold) and ATP modestly (ϳ3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.Walker A/B nucleotide binding motifs are conserved in all members of the RecA family, which includes hRAD51 1 and several other human homologs (1, 2). RecA and its homologs belong to a larger more distantly related family of proteins that also contain these motifs and whose biological function is mediated by nucleotide binding and hydrolysis. These include myosin, dynein, the F 0 /F 1 -ATPase/synthase, adenylate kinase, topoisomerase II, the MutS homologs, and G-proteins such as Ras and elongation factor Tu as well as others (1, 3, 4). Nucleotide-provoked conformational transitions are a hallmark of these proteins. It has been suggested that these proteins may function as switches where the NTP-and NDP-bound states reflect distinct conformational and biologically active states (1, 3, 4). These proteins utilize a variety of mechanisms to facilitate the NTP hydrolysis cycle and the transition between biologically relevant conformational states. For example, G-proteins may require additional protein factors that provoke ␥-phosphate hydrolysis (GTPase activator proteins) as well as the exchange of GDP for GTP (guanine nucleotide exchange factors) (5, 6). Other proteins, such as the F 1 -ATPase or the T7 gene 4 helicase/primase, utilize cooperative interactions between neighboring protomers to similarly facilitate NTP hydrolysis (7-11). In the case of the F 1 -ATPase, ADP release appears limiting and governed by the extent of cooperative ATP binding between ␣ and ␤ subunits (7, 9 -11).Cooperative ATP-induced conformational transitions are essential for RecA to assume and maintain an active nucleoprotein filament (12, 13). The first step in this process, ATP binding, results in conformational transitions that precede hydrolysis (14, 15). These transitions correlate with an extended nucleoprotein filament and appear to prevent microscopic reversibility during ATP hydrolysis (16). In addition, an actively hydrolyzing nucleoprotein filament appears to remain in an extended ATP-induced conformation (17). Moreover, ADP inhibition of the RecA ATPase displays hysteresis, suggesting that an actively hydrolyzing RecA nucleoprotein filament is not in continuous equilibrium (18). Taken together, these observations are consistent with the notion that cooperativity allows RecA to sustain a threshold of ATP-bound protomers within the nucleoprotein filament (12,13). Cooperativity between ATPbound protomers increases the efficiency of RecA mediated ATP hydrolysis, which in turn enhances the rate of recombinational strand exchange and allows the bypass of heterologous .DNA substantially stimulates the RecA ATPase (24, 25). DNA binding also correlates with an increased affinity of RecA for ATP and a decreased affinity of RecA for ADP (26,27). Thus, it ...

Ca 2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

“…Therefore, we concluded that accumulation of the inactive hRad51-ADP-ssDNA filament was caused by slow dissociation of ADP, the product of ATP hydrolysis. This conclusion is in accord with the report of Fishel and coworkers (35) that ADP dissociation is the rate-limiting step of ATP hydrolysis by hRad51 protein. In contrast, in the presence of Ca 2ϩ , the filament remains in an active hRad51-ATP-ssDNA form; the amount of protein-bound ADP did not exceed 3-4% after 30 min of incubation (Fig.…”

hXRCC2 Enhances ADP/ATP Processing and Strand Exchange by hRAD51

“…1E). In agreement with previous studies, we observed strong cross-linking of ATP to hRAD51 (21) and negligible cross-linking of ATP to hXRCC2 (Fig. 1E).…”

“…hRAD51 (1 M) was incubated at 37°C for 60 min in the presence or absence of hXRCC2 (1 M) or DNA (6 M nt or bp) with the indicated amount of ADP. Bound ADP was determined as described previously (21). ϫ, hXRCC2 in the presence or absence of DNA; E, hRAD51 alone; Ⅺ, hRAD51 alone with X174 ssDNA; ‚, hRAD51 alone with X174 dsDNA RFI; q, hRAD51 plus hXRCC2; f, hRAD51 plus hXRCC2 with X174 ssDNA; OE, hRAD51 plus hXRCC2 with X174 dsDNA RFI.…”

“…UV Cross-linking-Reactions were performed as described previously (21). In brief, 1 M hXRCC2 and 1 M hRAD51 were incubated at 25°C for 15 min.…”

“…1F; Table I). The low affinity mode has been shown to correlate with a nonspecific hRAD51-DNA aggregate (21,22). Co-incubation of hRAD51 with hXRCC2 completely suppressed the low affinity binding of ADP by hRAD51 ( Fig.…”