Biochemical Characterization of the Human RAD51 Protein

Tombline, Gregory; Heinen, Christopher D.; Shim, Kyung Won; Fishel, Richard

doi:10.1074/jbc.m109917200

Cited by 40 publications

(17 citation statements)

References 49 publications

(59 reference statements)

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“…Moreover, ADP 3 ATP exchange appears to depend on the extent of ADP saturation. These and additional studies presented in the following paper hRAD51 Nucleotide Binding/Exchange (37) are consistent with the notion that elevated ADP induces an irreversible aggregate structure that may only be relevant to in vitro studies.…”

Section: Discussionsupporting

confidence: 67%

Biochemical Characterization of the Human RAD51 Protein

Tombline

Shim

Fishel³

2002

Journal of Biological Chemistry

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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 ...

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Section: Discussionsupporting

confidence: 67%

Biochemical Characterization of the Human RAD51 Protein

Tombline

Shim

Fishel³

2002

Journal of Biological Chemistry

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“…Unlike bacterial RecA protein, yeast or human Rad51 readily bind dsDNA [118, 153]. This biochemical property poses a significant problem for the cell, as Rad51 may form dead-end complexes on chromosomal DNA, which exists in vast excess over sites of DNA damage, where Rad51 is destined to assemble on ssDNA [117] (Fig.…”

Section: Functions Of Rad54 Proteinmentioning

confidence: 99%

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Ceballos

Heyer

2011

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

130

141

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Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis.

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“…Although hRad51 shares many structural and functional similarities with RecA and the general process of recombination is similar for both, it differs significantly from RecA in certain biochemical characteristics. RecA has a very strong preference for nucleation on ssDNA ( 4 , 5 , 17 ) and ATP hydrolysis promotes its dissociation from ssDNA and dsDNA ( 17–20 ), whereas hRad51 shows a preferential affinity for dsDNA ( 21 ), it has a very low ATPase activity ( 6 ) and ATP hydrolysis does not seem to correlate with hRad51 dissociation from dsDNA ( 22 , 23 ). Since efficient strand exchange requires preferential binding to ssDNA rather than dsDNA ( 7 , 17 ), this might explain hRad51's weaker strand-exchange activity in vitro when compared to RecA, and suggests that one of the roles of the numerous other proteins involved in HR in vivo may be to favour formation of hRad51–ssDNA nucleoprotein filaments over dsDNA ones.…”

Section: Introductionmentioning

confidence: 99%

Real-time measurements of the nucleation, growth and dissociation of single Rad51–DNA nucleoprotein filaments

Miné

Disseau

Takahashi

et al. 2007

Nucleic Acids Research

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Human Rad51 (hRad51), the protein central to DNA pairing and strand exchange during homologous recombination, polymerizes on DNA to form nucleoprotein filaments. By making use of magnetic tweezers to manipulate individual DNA molecules, we measured the nucleation and growth of hRad51 nucleoprotein filaments, and their subsequent disassembly in real time. The dependence of the initial polymerization rate upon the concentration of hRad51 suggests that the rate-limiting step is the formation of a nucleus involving 5.5 ± 1.5 hRad51 monomers, corresponding to one helical turn of the hRad51 nucleoprotein filament. Polymerization is highly cooperative (i.e. a nucleation-limited reaction) at low concentrations and less cooperative (a growth-limited reaction) at high concentrations of the protein. We show that the observed preference of hRad51 to form nucleoprotein filaments on double-stranded DNA rather than on single-stranded DNA is due to the fact that it depolymerizes much faster from ssDNA than from dsDNA: indeed, hRad51 polymerizes faster on ssDNA than on dsDNA. Hydrolysis of ATP by hRad51 does not correlate with its dissociation from dsDNA. This suggests that hRad51 does not depolymerize rapidly from dsDNA after strand exchange but stays bound to the heteroduplex, highlighting the importance of partner proteins to facilitate hRad51 depolymerization from dsDNA.

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Biochemical Characterization of the Human RAD51 Protein

Cited by 40 publications

References 49 publications

Biochemical Characterization of the Human RAD51 Protein

Biochemical Characterization of the Human RAD51 Protein

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Real-time measurements of the nucleation, growth and dissociation of single Rad51–DNA nucleoprotein filaments

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