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J. Biol. Chem., Vol. 277, Issue 17, 14434-14442, April 26, 2002

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Biochemical Characterization of the Human RAD51 Protein

III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES^*

Gregory Tombline, Christopher D. Heinen, Kang-Sup Shim, and Richard Fishel

From the Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, October 12, 2001, and in revised form, February 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adenosine nucleotides affect the ability of RecA·single-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51·ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA·ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51·ssDNA binding form (hRAD51·ssDNA_low) correlates with active ADP and ATP processing. A high molecular weight hRAD51·ssDNA aggregate (hRAD51·ssDNA_high) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adenosine nucleotide binding and hydrolysis by the bacterial RecA protein is crucial to mediate the formation, stability, and structure of RecA·ssDNA¹ (nucleoprotein) filament thought to be the key intermediate in the initiation of recombination in vitro and in vivo (1-3). Although hRAD51 is a homolog of RecA, the extent to which adenosine nucleotides may modulate the formation, structure, and stability of hRAD51 nucleoprotein filaments is unknown.

RecA couples the binding of ADP or ATP/ATPS with the binding to DNA. In the presence of ATPS, RecA binds to ssDNA in a highly cooperative, largely irreversible manner and extends the helical pitch of the DNA to ~1.5 times its normal length (4-7). Reciprocally, ssDNA increases the affinity of RecA for ATPS (8). In the presence of ADP, RecA binding to ssDNA is unstable, and the filament is less extended/inactive (4-6). Similarly, ssDNA decreases the affinity of RecA for ADP (9). In contrast, both ssDNA and dsDNA failed to affect ATPS binding or ADP release by hRAD51 (10). Moreover, hRAD51 appears to lack ATP-induced cooperativity during ATP hydrolysis (11) and/or the ability to form extended/active hRAD51·ssDNA nucleoprotein filaments in the presence of ATPS (12). These data suggest that hRAD51 differs significantly from RecA in its ability to couple ADP and ATP/ATPS processing with ssDNA interactions.

The ability of RecA to self-associate in the absence of DNA adds to the complexity of RecA-ssDNA interactions. Several methods, including gel filtration chromatography, electron microscopy, light scattering, and analytical ultracentrifugation, have demonstrated that in the absence of DNA, RecA exists as dimers, trimers, small planar hexameric-to-octameric rings, and short rods (13-15). Larger rods/filaments and bundles of filaments form in the presence of cations (magnesium or spermidine) or concentrated solutions of RecA (16). While the ability to self-associate appears to facilitate cooperative RecA nucleoprotein filament assembly (17), the exact species of RecA that binds to or dissociates from DNA is unknown. Moreover, beyond a certain threshold of RecA self-association, DNA binding is significantly reduced (18). Efficient DNA binding by RecA appears to involve prior disassembly of self-aggregates to a relatively smaller species (18-20). Both ATP and ADP reduce the size of self-aggregates (14, 16), generating polymers ranging from hexamers to dodecamers in size (13). However, only ATP (ATPS) efficiently activates RecA DNA binding activity (4-6). Little is known about the effect of ADP, ATP, or ATPS on the conformation of hRAD51, its ability to form self-polymers, or its ability to assume and maintain an active hRAD51·ssDNA nucleoprotein filament.

Predicting the coordinate self-association, nucleotide binding/hydrolysis, and ssDNA binding activity of hRAD51 based on a comparison with the RecA protein structure is not straightforward. The RecA crystal structure indicated that residues involved in self-association, adenosine nucleotide binding/hydrolysis, and DNA binding appeared juxtaposed and coordinated (21, 22). Genetic studies indicated that RecA residues His⁹⁷, Lys²¹⁶, Phe²¹⁷, Arg²²², and Lys²⁴⁸ are involved in monomer-monomer contacts and are also proximal to the ADP binding pocket as well as the unresolved loops implicated in DNA binding (23, 24). It has been proposed that RecA residues Gln¹⁹⁴ and Arg¹⁹⁶ interact with the -phosphate of ATP to coordinate DNA binding by loop L2 (residues 195-210) with ATP hydrolysis (25, 26). Moreover, Hörtnagel et al. (26) suggested that Arg¹⁹⁶ may participate catalytically in ATP hydrolysis in a manner similar to the "arginine finger" of GTPase activator proteins (27). These residues appear critical for biological activity and probably function as allosteric effectors of the ATP-induced active form of the RecA nucleoprotein filament (23, 24).

Although these residues are highly conserved in the eubacterial RecA family, an entirely different set of residues may substitute for their functions in the eukaryotic RAD51 members (28). Only Arg²²² and Gln¹⁹⁴ appear conserved between RecA and hRAD51. In addition, hRAD51 protein has an extended amino terminus and truncated carboxyl terminus relative to the bacterial RecA (28, 29). The amino terminus of RecA forms extensive contacts between neighboring protomers within a filament and is critical for self-association (30), while the carboxyl terminus of RecA exists as a distinct domain that appears to modulate DNA binding (31, 32). Interestingly, a study that combined NMR and mutagenesis indicated that the extended amino terminus of hRAD51 may functionally replace the carboxyl-terminal domain of RecA (33).

Despite these differences, preliminary electron microscopy data have indicated that hRAD51 forms planar hexameric-to-octameric ring structures in the absence of DNA (34). In addition, nucleoprotein filaments formed in the presence of ATPS with ssDNA grossly resemble inactive/compact RecA nucleoprotein filaments (12). In contrast, neutron scattering performed on Xenopus laevis RAD51 suggested that an extended filament may form on ssDNA in the presence of ATP or ADP but less well with ATPS (35). Interestingly, hRAD51 forms an extended nucleoprotein filament with the transition state mimetic ADP-AlF, which appears analogous to activated RecA (36). Both RecA and hRAD51 facilitate ATP-dependent DNA strand exchange. However, unlike RecA, hRAD51 could not utilize ATPS for DNA strand exchange (37).

Here we have examined the effects of ADP, ATP, and ATPS on hRAD51 ssDNA binding activity. We find significant differences in the effects of these adenosine nucleotides on the ssDNA binding activity of hRAD51 compared with RecA. Unlike RecA, hRAD51 binding of ADP, ATP, or ATPS is not coupled to modulation of ssDNA binding activity. In addition, hRAD51 appears largely unable to discriminate ADP, ATP, or ATPS. It is likely that the inability of RAD51 to efficiently process adenosine nucleotides underpins the lack of coordination between protomers during ATP hydrolysis. We propose that inefficient ATP processing contributes to a reduced rate of DNA strand exchange and a dramatically reduced ability to bypass heterologous DNA during strand exchange (38-41).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- Chemicals of the highest grade were obtained from Amresco or Sigma. ADP and ATP were purchased from Amersham Biosciences and processed as described (11). ATPS was purchased from Roche Molecular Biochemicals. Sequencing grade endoprotease Lys-C and trypsin were purchased from Promega or Roche Molecular Biochemicals and resuspended in modified H buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 10% glycerol, without EDTA) at a stock concentration of 200 ng/µl just prior to use. Oligo(dT)₅₀ was synthesized at the Kimmel Nucleic Acids Facility. IAsys cuvettes coated with biotin were purchased from Affinity Sensors (Cambridge, UK), and 5' or 3' biotinylated oligo(dT)₅₀ was purchased from Glenn Research (Sterling, VA). hRAD51 was purified as described (11). RecA protein was purchased from U.S. Biochemical Corp.

Partial Proteolysis-- Approximately 10 µg of hRAD51 was incubated in the standard buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA) in 50 µl with 10 mM magnesium acetate and 1 mM ATP, ADP, or ATPS at 37 °C for 30 min in an Ericomp thermal cycler with heated lid to prevent evaporation. Then 6-µl aliquots were added to 6 µl of various protease dilutions. Shown are digests containing either 220 ng of endoprotease Lys-C or 220 ng of trypsin. Protease incubations continued at 37 °C for an additional 60 min. Reactions were stopped by the addition of 4 µl of 4× loading buffer (0.2 M Tris-HCl (pH 7.0), 8% SDS, 20% -mercaptoethanol, 20% sucrose, 2 mg/ml bromophenol blue) and followed by boiling. Proteolytic products of hRAD51 were resolved by 20% SDS-PAGE and silver-stained. Gels were wrapped in clear plastic wrap and scanned directly with an Epson scanner.

Analytical Gel Filtration-- 100-200 µl of 8 µM hRAD51 was preincubated at 37 °C for 60 min in modified H buffer with 2 mM magnesium acetate without nucleoside or with 20 µM ATPS or 20 µM ADP. A Superose-12 column (10/30; Amersham Biosciences) was equilibrated in the same buffer. hRAD51 was eluted at a flow rate of 24 ml/h, and 0.5-ml fractions were collected. 1 µg of acetylated bovine serum albumin (New England Biolabs) and 0.5 ml of 1 M trichloroacetic acid were added to each fraction, followed by overnight precipitation at 20 °C. The precipitated proteins were then pelleted, washed with ice-cold acetone, air-dried, and resuspended in loading buffer (0.05 M Tris-HCl (pH 7.0), 2% SDS, 5% -mercaptoethanol, 5% sucrose, 0.5 mg/ml bromophenol blue). Approximately half of the precipitate was resolved by 10% SDS-PAGE and silver-stained.

IAsys Biosensor Studies-- IAsys biosensor (IAB) studies were performed using an IAsys Auto+ unit (Affinity Sensors, Cambridge, UK). A model oligonucleotide (oligo(dT)₅₀) with either the 3'-end or 5'-end biotinylated (Glen Research; Sterling, VA) was attached via streptavidin to the surface of an IAsys SPR cuvette precoated with biotin (Affinity Sensors). The kinetics of hRAD51 DNA binding were measured after the cuvettes were equilibrated in H buffer containing the indicated amount of magnesium. The binding constant, k_a, was obtained directly from the slope of a plot of the k_on determined at multiple hRAD51 protein concentrations. Dividing the k_d, determined experimentally via IAB and averaged from several protein concentrations, by the k_a results in the K_D (K_D = k_d/k_a). This method was also used by DeZutter et al. (42) to determine the K_D for hRAD51 binding to an 83-mer random sequence oligonucleotide.

Gel Shifts-- Oligo(dT)₅₀ binding was performed in H buffer with 250 nM (nucleotide equivalents) of oligo(dT)₅₀ and the indicated amount of hRAD51. Reactions were incubated as indicated in each figure legend and resolved by 4% nondenaturing PAGE (acrylamide/bisacrylamide, 37.5:1; Amresco) containing 0.5% glycerol. The system was buffered with 1× TBE, and 30-mA constant current was applied to each gel for ~2.5-3 h. The gels were placed on Whatman No. 3MM paper, exposed directly (without drying) to PhosphorImager screens (Molecular Dynamics, Inc., Sunnyvale, CA), and scanned after overnight exposure. Care was taken not to disrupt hRAD51·DNA aggregates remaining in the wells. We found that if the gel is sufficiently wrapped to avoid contamination of the PhosphorImager screen, it may be exposed without drying, and all of the aggregate species will be retained.

Sedimentation Assay-- hRAD51 (0.5 µM) was incubated with 5' end- ³²P-labeled oligo(dT)₅₀ (50 nM nucleotide equivalents) in 20 µl for 60 min at 37 °C in the standard H buffer with 10 mM magnesium acetate in the absence of nucleotide or with either 20 or 500 µM of each adenosine nucleotide added, as indicated in the figure legend. Each reaction was centrifuged in a microcentrifuge at 14,000 rpm (~16,000 × g) for 30 min. The supernatant (19 µl) was removed carefully and mixed with 5 ml of Scintiverse mixture (Fisher) and counted. To the pellet, 100 µl of 10% SDS was added, and the sample was then vortexed, boiled, mixed with 5 ml of Scintiverse, and counted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA Binding by hRAD51

IAsys Biosensor Studies-- IAB is capable of detecting real time interactions between molecules. We have developed a system in which a model oligonucleotide (oligo(dT)₅₀) is biotinylated at either the 3'- or 5'-end and attached via a streptavidin linkage to an IAB cuvette coated with biotin.

We obtained K_D values for hRAD51·oligo(dT)₅₀ binding in the range of 78-176 nM depending upon the magnesium concentration or whether the 5'- or 3'-biotinylated substrate was examined (Table I). These results largely resemble a previous study that measured the interaction of hRAD51 with ssDNA (a random sequence 83-mer oligonucleotide) by IAB (42). Modest differences between these studies may be attributed to the size and/or nature of the secondary structure present in the two substrate DNAs or the different buffer conditions. We observed that 3'-biotinylated oligo(dT)₅₀ (3'-end attached to the IAB cuvette surface) consistently displayed a lower K_D that could be largely attributed to an increased k_a. These results suggest that hRAD51 can bind ssDNA displaying either a free 5'-end or 3'-end but that it exhibited a modest preference for binding DNA displaying a free 5'-end.

hRAD51 (maintained at 4 °C) readily bound the oligo(dT)₅₀ IAB DNA substrate in the absence of adenosine nucleotide as well as in the presence of ADP or ATP (B_max  1000 arc s for each; Fig. 1A). Moderately less efficient binding was observed in the presence of ATPS (B_max  500 arc s; Fig. 1A). When hRAD51 was preincubated at 37 °C for 30 min in the absence of adenosine nucleotide, it failed to bind the oligo(dT)₅₀ IAB DNA substrate (B_max < 100 arc s; Fig. 1, compare A and C). However, when ADP, ATP, or ATPS was present during the preincubation, binding to the oligo(dT)₅₀ IAB DNA substrate by hRAD51 preincubated at 37 °C appeared almost identical to the binding displayed by hRAD51, which was maintained at 4 °C (B_max  1000 arc s; Fig. 1, compare A and C). These observations suggest that incubation of hRAD51 at 37 °C results in a form that is largely incapable of ssDNA binding.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Comparative effects of adenosine nucleotides on ssDNA binding by hRAD51 and RecA. IAsys biosensor analysis was used to compare the binding of 125 nM hRAD51 (A and C) or 250 nM RecA (B and D) to a model ssDNA substrate (oligo(dT)50) in the presence of 10 mM Mg2+ and in the absence or presence of 1 mM ADP, ATP, or ATPS. Prior to the addition of proteins, IAsys cuvettes with covalently attached biotin were precoated with streptavidin followed by oligo(dT)50 containing a 5' biotin. In A and B, the protein/nucleotide mix was added to the cuvette directly, whereas in C and D, each protein/nucleotide mix was preincubated at 37 °C for 30 min prior to the addition to the cuvette.

We compared the binding of the oligo(dT)₅₀ IAB DNA substrate by hRAD51 to the binding of this substrate by RecA. ATPS promoted the most stable interaction of RecA with the oligo(dT)₅₀ IAB DNA substrate (B_max  900 arc s; Fig. 2B). RecA binding in the absence of adenosine nucleotide was slightly less efficient (B_max  500 arc s; Fig. 1B). Under our conditions (150 mM NaCl), RecA failed to bind the oligo(dT)₅₀ IAB DNA substrate in the presence of ATP or ADP (B_max < 100 arc s; Fig. 1B). These results are qualitatively and quantitatively similar to previously published reports (4-7, 42). Moreover, RecA binding appeared identical regardless of the preincubation conditions (Fig. 1, compare B with D). These observations underline the inherent differences in ssDNA binding between RecA and hRAD51.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Adenosine nucleotides preserve hRAD51·oligo(dT)50 binding activity. Reactions were performed as in Fig. 1C except that the indicated amount of ATP (A) or ADP (B) was substituted during the 30-min preincubation period. Each point represents the maximal bound. Each bar represents the average of three experiments.

We determined the amount of ADP (IC₅₀  1 µM) or ATP (IC₅₀  0.2 µM) required to preserve half-maximal hRAD51·oligo(dT)₅₀ IAB DNA binding activity (Fig. 2). The IC₅₀ values appear significantly less than the binding constant determined for ADP or ATPS to hRAD51 (K_D  5 µM) (10). These data appear to suggest that only a subset of hRAD51 protomers must be bound by ADP or ATP to preserve DNA binding activity. AMP did not significantly preserve DNA binding (data not shown), suggesting that the -phosphate of ADP is minimally required to preserve oligo(dT)₅₀ IAB DNA binding activity. These observations are consistent with a significantly reduced affinity of hRAD51 for AMP (10).

Gel Shift Studies-- The effect of ADP, ATP, or ATPS on the interaction of hRAD51 with oligo(dT)₅₀ was also examined by gel shift analysis. hRAD51 appears to form at least two qualitatively different forms when bound to the model oligo(dT)₅₀ ssDNA substrate, which we denote hRAD51·DNA_low and hRAD51·DNA_high (Fig. 3, A and B). Moreover, the migration of the hRAD51 forms appears distinct from RecA·oligo(dT)₅₀ gel shift complexes produced under identical conditions (compare Fig. 3, A and B, with Fig. 3C). The structure of these forms is unknown. The hRAD51·DNA_low form appears be a low molecular weight complex that migrates slightly more slowly than free oligo(dT)₅₀ (denoted by an asterisk; Fig. 3, A and B). The hRAD51·DNA_high form is a significantly larger complex (Fig. 3, A and B). The relative amount of these forms appears to be modulated by ADP, ATP, and ATPS. In the absence of ADP, ATP, and ATPS, hRAD51·DNA_low is the singular gel shift form (Fig. 3A, lanes 1-5). In contrast, the addition of 1 mM ADP, ATP, and ATPS induces the formation of hRAD51·DNA_high (Fig. 3A, lanes 6-10). The relative amount of the hRAD51·DNA_high form depends on the type of adenosine nucleotide, where ADP > ATPS ATP (Fig. 3A, lanes 6-10 > lanes 16-20 lanes 11-15). By quantitating the ratio of free DNA to bound DNA (where bound represents both forms), we calculate a K_D(hRAD51)  0.3 µM for binding to the oligo(dT)₅₀ ssDNA substrate in the presence of ADP or ATP or in the absence of adenosine nucleotide (Fig. 3A, lanes 6-10, lanes 11-15, or lanes 1-5). In the presence of ATPS, hRAD51 displayed a reduced affinity for the oligo(dT)₅₀ ssDNA substrate (K_D(hRAD51)  0.5 µM; Fig. 3A, lanes 16-20).

View larger version (67K): [in this window] [in a new window]   Fig. 3.   Gel shift analysis of hRAD51 and RecA binding to oligo(dT)50. A, the indicated amount of hRAD51 (µM) was incubated with oligo(dT)50 (250 nM) for 60 min at 37 °C in the absence or presence of 1 mM ADP, ATP, or ATPS and then resolved by 4% nondenaturing PAGE. B, reactions are identical to A except that hRAD51 was preincubated in the absence or presence of adenosine nucleotide for 30 min at 37 °C and subsequently mixed with oligo(dT)50. These were incubated at 37 °C for an additional 20 min and then resolved by 4% nondenaturing PAGE. C, reactions were identical to A except that the indicated amount of RecA (µM) was substituted for hRAD51. Two different hRAD51·oligo(dT)50 complexes are indicated by low and high; an asterisk indicates free oligo(dT)50. N, reactions lacking hRAD51 or RecA.

Gel shift analysis following preincubation of hRAD51 at 37 °C in the absence or presence of ADP, ATP, or ATPS also resulted in a DNA binding pattern qualitatively similar to IAB (compare Fig. 1, A and C, with Fig. 3, A and B). In the absence of adenosine nucleotide, hRAD51 appeared to be largely inactivated, since DNA binding was only observed at very high concentrations of hRAD51 (Fig. 3B, lanes 1-5). Under these conditions, the K_D(hRAD51) did not appear to change when nucleotide was included (similar to IAB). However, a significant increase in the hRAD51·DNA_high form and coincident decrease in the hRAD51·DNA_low form occurred in the presence of ADP (Fig. 3B, lanes 6-10). A similar but less striking effect occurred in the presence of ATPS (Fig. 3B, lanes 16-20). In the presence of ATP, the distribution between forms appeared to remain unchanged (Fig. 3B, lanes 11-15).

We examined the effect of ADP, ATP, and ATPS on hRAD51 binding to the oligo(dT)₅₀ ssDNA substrate by gel shift analysis. These studies were performed at a concentration of hRAD51 (0.3 µM) that largely generated the hRAD51·DNA_low form and minimized the production of aggregates (hRAD51·DNA_high). We found that ADP, ATP, or ATPS enhanced the formation of hRAD51·DNA_low (K_D(NUC)  1 µM; Fig. 4, A-C). Above 250 µM ADP, ATP, or ATPS, the effect of these nucleotides appears more complex (Fig. 4). ADP promotes the production of the hRAD51·DNA_high form (Fig. 4A). ATP (Fig. 4B) and ATPS (Fig. 4C) promote the dissociation of a fraction of the hRAD51·DNA_low form. Both gel shift analysis and IAB confirm the apparent equivalent high affinity of adenosine nucleotides for hRAD51. These results also suggest that the type of hRAD51·ssDNA form generated is influenced by adenosine nucleotides. Interestingly, dsDNA does not appear to form hRAD51·DNA_high structures similar to ssDNA (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Saturation of the hRAD51 high affinity adenosine nucleotide binding site (KD  5 µM) enhances the formation of the hRAD51·DNAlow species. hRAD51 (0.3 µM) was mixed with the indicated amount of each adenosine nucleotide and 250 nM oligo(dT)50 (nucleotides), incubated at 37 °C for 30 min, and subsequently resolved by 4% nondenaturing PAGE. An asterisk indicates the position of free oligo(dT)50 DNA.

Our previous studies have demonstrated that binding of ADP to hRAD51 displayed two distinct modes: one mode with a high affinity for ADP (K_app1  5 µM), which appeared competitive with ATP, and a second low affinity mode (K_app2  125 µM) (10). The first mode (K_app1  5 µM) appeared to be competent for ADP  ATP exchange with a t_1/2  30 s, whereas the second mode (K_app2  125 µM) appeared refractory for ADP  ATP exchange (10). We found that the formation of hRAD51·DNA_high correlated well with the expected K_app2  125 µM (Fig. 5A). Furthermore, there appears to be a transition from hRAD51·DNA_low to hRAD51·DNA_high in our gel shift analysis with increasing ADP saturation (Fig. 5A). When 5 mM ATPS was added subsequent to ADP in these DNA-binding reactions, the hRAD51·DNA_low form dissociated, while the hRAD51·DNA_high form remained unchanged (Fig. 5, compare A and B). Moreover, once formed, hRAD51·DNA_high aggregate appears stable and irreversible (data not shown). Taken together, these results demonstrate that ADP, ATP, and ATPS modulate the hRAD51·DNA_low form. However, the hRAD51·DNA_high form is likely to be a "dead end" complex that may only be significant for in vitro assays.

View larger version (66K): [in this window] [in a new window]   Fig. 5.   Adenosine nucleotides affect the formation and stability of hRAD51·DNAlow and hRAD51·DNAhigh complexes. A, hRAD51 (0.5 µM), the indicated amount of ADP, and 250 nM oligo(dT)50 (nucleotides) were incubated at 37 °C for 60 min and then resolved by 4% nondenaturing PAGE. B, reactions were performed as in A except that 5 mM ATPS was added subsequent to the initial 60-min incubation, and the incubation was continued for an additional 20 min at 37 °C prior to PAGE. C and D, reactions were performed as in B except that the initial incubation contained 20 µM ADP, and the indicated amount of ATPS (C) or ATP (D) was added subsequently. An asterisk indicates the position of free oligo(dT)50 DNA. For C and D, the relative species denoted by an asterisk and low were quantitated by a PhosphorImager, and the data were fit to a curve by nonlinear regression. The resulting curves are displayed below their respective gels.

The apparent replacement of ADP by ATPS that resulted in the destabilization of hRAD51·DNA_low occurred with a K_i·ATPS  100 µM and translated to a ratio of 1 ADP/4-5 ATPS (Fig. 5C). ATP appeared significantly less effective in destabilizing the hRAD51·DNA_low form (K_i·ATP  250 µM; Fig. 5D). The amount of ATP (ATPS) required to destabilize the hRAD51·DNA_low form generated in the presence of ADP is similar to that required for the destabilization of hRAD51·DNA_low in the absence of ADP (Fig. 4, B and C). These results suggest that a specific ratio of ADP/ATP (ATPS) may not be required for efficient dissociation.

Sedimentation Assay-- The RecA protein has been found to form aggregates and co-aggregates with ssDNA and dsDNA. The RecA/DNA co-aggregates appeared to be intermediates in the homologous pairing process and could be easily pelleted (43). Although a comparison between these structures and the hRAD51·DNA_high aggregates would be premature, we reasoned that a similar co-sedimentation assay in the presence of ADP, ATP, or ATPS would be useful for the analysis of the hRAD51·DNA_high form. The addition of hRAD51 resulted in co-sedimentation of the ³²P-labeled oligo(dT)₅₀ ssDNA substrate in the presence of 500 µM ADP (Fig. 6B). No co-sedimentation was observed in the absence of adenosine nucleotide (Fig. 6A), in the presence of 20 µM ADP (Fig. 6B), or in the presence of either 20 or 500 µM ATP/ATPS (Fig. 6, C and D). These results are consistent with the hRAD51 oligo(dT)₅₀ ssDNA gel shift analysis and suggest that hRAD51 forms a hRAD51-ssDNA aggregate at supersaturating concentrations of ADP.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   hRAD51 and oligo(dT)50 form large complexes in the presence of high ADP concentrations. To further characterize hRAD51·oligo(dT)50 complexes, the ability to co-sediment in the absence or presence of adenosine nucleotides was examined. hRAD51 (5-500 nM) and oligo(dT)50 (50 nM) were incubated at 37 °C for 60 min in the absence of nucleotide (A) or in the presence of 20 or 500 µM ADP (B), ATP (C), or ATPS (D). After centrifugation at 16,000 × g for 30 min at room temperature, counts retained in the supernatant (sup) and pellet were determined. Each point represents the average of three experiments.

Adenosine Nucleotides Induce Conformational Transitions in hRAD51

Partial proteolysis has been a useful method for determining conformational transitions associated with nucleotide-binding proteins, including RecA (44). hRAD51 was preincubated with ADP, ATP, or ATPS and subsequently exposed to a limiting amount of either endoprotease Lys-C or trypsin. Partial proteolysis by both endoprotease Lys-C and trypsin suggest that ADP, ATP, and ATPS induced conformational transitions in hRAD51 that were distinct from the pattern exhibited in the absence of adenosine nucleotide (Fig. 7, A and B). Endoprotease Lys-C generates two distinct peptides (denoted by arrows in Fig. 7A) of ~15-20 kDa that were evident when hRAD51 was incubated with buffer only but not detectable in the presence of ADP, ATP, or ATPS. Partial trypsin digestion generated two distinct peptides of ~25-30 kDa (see arrows in Fig. 7B) that were prominent when ADP, ATP, or ATPS was added but not detectable in the presence of buffer alone. The specific location of these peptides within the hRAD51 protein is currently under investigation. The appearance of these partial proteolysis peptides corresponded with the ADP, ATP, and ATPS binding affinity (K_D  5 µM; data not shown), and the peptide banding pattern did not appear to differ between adenosine nucleotides. In the absence of protease only full-length hRAD51 was observed, and in the absence of hRAD51 there were no peptide products (data not shown). These results indicate that ADP, ATP, or ATPS affects the susceptibility of hRAD51 to protease, suggesting alternate conformations.

View larger version (82K): [in this window] [in a new window]   Fig. 7.   Adenosine nucleotides affect hRAD51 sensitivity to limited proteolysis and the ability to self-associate. Limited proteolysis (A and B) and analytical gel filtration (C) were performed to examine the effects of adenosine nucleotides on hRAD51 structure. For proteolysis, ~10 µg of hRAD51 was preincubated at 37 °C for 30 min in the absence or presence of a 1 mM concentration of the indicated adenosine nucleotide. These were subsequently divided into two 5-µg portions and treated with either endoproteinase Lys-C (A) or sequencing grade trypsin (B). Proteolysis products were resolved by 20% SDS-PAGE followed by silver staining. C, hRAD51 (8 µM) was preincubated at 37 °C for 60 min in the absence or presence of 20 µM ADP or ATPS and subsequently resolved by elution through a Superose 12 column equilibrated with the same buffer. Each fraction was precipitated with trichloroacetic acid and analyzed by 10% SDS-PAGE followed by silver staining. Relative molecular weights were determined with gel filtration standards (Bio-Rad) and are indicated above the fraction numbers.

Adenosine Nucleotides Affect hRAD51 Self-association

Analytical gel filtration chromatography has been used to determine higher order protomer complexes of the bacterial RecA protein (15). In the absence of adenosine nucleotides, analytical gel filtration at 4 °C suggests that hRAD51 self-associates to form a protein species that appears to migrate with the relative molecular weight of hexamers to dodecamers (data not shown). These protein species may resemble the hRAD51 hexameric/octameric rings observed previously by electron microscopy (34). In contrast, incubation of hRAD51 at 37 °C in the absence of adenosine nucleotide results in an apparent high molecular weight species of hRAD51 (Fig. 7C). A fraction of these complexes are found in the void volume of the gel filtration column that was determined to exclude proteins of ~1 MDa. This result suggests that at 37 °C in the absence of adenosine nucleotide, hRAD51 forms higher order self-association complexes similar to RecA. The detailed structure of these complexes is unknown. It is interesting to note that our chromatography buffer contained 150 mM NaCl, which is inhibitory to the self-association/polymerization of RecA (15). The ability of hRAD51 to aggregate in the presence of physiological salt concentration appears similar to C-terminally truncated mutants of RecA (45) and X. laevis RAD51 (35). These results suggest that hRAD51 may possess an increased intrinsic potential to aggregate.

Chromatography of hRAD51 in the presence of either 20 µM ADP or ATPS results in protein species that appear to elute with an average molecular mass of 100-250 kDa, corresponding to 3-8 hRAD51 monomers (Fig. 7C). These results suggest that the addition of ADP or ATPS significantly reduced the size of the nucleotide-free hRAD51 aggregates. The amount of ADP or ATPS required for this reduction correlated with the previously identified hRAD51 high affinity binding mode (K_D  5 µM; Fig. 7C and Ref. 10). These results suggest that both ADP and ATP are capable of modulating the size of hRAD51 polymers.

	DISCUSSION

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Bacterial RecA protein cooperatively binds and hydrolyzes ATP in a process that is intrinsically linked to the structure/stability of a RecA·ssDNA nucleoprotein filament (1, 3). These observations have been described in terms of a "coupled cycle." Several studies have shown that a tight association exists between ATP/ATPS-bound RecA and ssDNA and a weak association exists between ADP-bound RecA and ssDNA (1, 3). Cooperative interactions between ATP-bound RecA protomers within this nucleoprotein filament appear to facilitate ADP  ATP exchange and drive the RecA ATPase cycle. The hRAD51 ATPase lacks the magnitude of ATP-induced cooperativity displayed by RecA (11). Moreover, ssDNA does not affect the affinity of hRAD51 for ADP or ATP/ATPS (10). These observations have suggested that hRAD51 may not couple adenosine nucleotide processing and DNA binding similar to RecA.

We have examined the effect of ADP, ATP, or ATPS on hRAD51 ssDNA binding by IAB as well as gel shift (GS) analysis. In the absence of adenosine nucleotide, hRAD51 exhibited an enhanced affinity for a model oligo(dT)₅₀ ssDNA substrate compared with RecA. The effect of ADP, ATP, and ATPS on hRAD51 ssDNA binding appeared nearly opposite to RecA. Unlike RecA, and consistent with a previous report, hRAD51 displayed reduced affinity for ssDNA in the presence of ATPS (42). We now report that hRAD51 ssDNA binding was unaffected by ADP and ATP. This contrasts with the reduced or absent RecA ssDNA binding activity in the presence of ADP and ATP (see Figs. 1 and 3) (4-7, 42). These results suggest a fundamental difference between RecA and hRAD51 in the effect of ADP, ATP, and ATPS on ssDNA binding functions.

A temperature-dependent inactivation of hRAD51 ssDNA binding activity was observed using both IAB and GS analysis. Saccharomyces cerevisiae RAD51 also exhibits a temperature-dependent inactivation of ssDNA binding activity (46). Maintaining the yeast RAD51 in the presence of ATP (K_D(ATP)  20 µM) or ADP (K_D(ADP) 250 µM) appeared to preserve the ssDNA binding activity (46). In contrast, hRAD51 required significantly less ATP (K_D(ATP)  0.1 µM) or ADP (K_app1  5.0 µM) to preserve ssDNA binding activity. The amount of ATP required to preserve hRAD51 ssDNA binding activity is 20-fold below the K_D(ATP). These data suggest that preservation of hRAD51 ssDNA binding activity at elevated temperatures does not require saturation of the protein with ATP. The difference between ATP and ADP preservation of hRAD51 ssDNA binding activity is unknown.

The temperature inactivation of hRAD51 ssDNA binding activity in the absence of ADP, ATP, or ATPS correlates with the appearance of a high molecular mass (670 kDa) aggregate by gel filtration (see Fig. 7C). In the presence of ADP and ATPS, hRAD51 is resolved into smaller complexes (~40-200 kDa) that correlate with ssDNA binding activity (see Fig. 7C). These results are consistent with the notion of self-aggregation of hRAD51 in the absence of adenosine nucleotide. An aggregation effect that is competitive with ssDNA binding has been observed with RecA (18, 19). In the case of hRAD51, the aggregate appears irreversible and is only likely to be relevant to in vitro assays. It is also theoretically possible that the ADP-dependent irreversible self-association/aggregation of hRAD51 may be modulated by other protein factors

GS analysis of hRAD51·ssDNA complexes identified two qualitatively different hRAD51·oligo(dT)₅₀ complexes: a fast mobility, apparently low molecular weight form (denoted hRAD51·DNA_low) and a slow mobility, apparently high molecular weight aggregate form (denoted hRAD51·DNA_high). While the precise nature of these forms is unknown, the mobilities of the hRAD51·ssDNA forms are distinct from the RecA·ssDNA complex. Since the molecular weights of RecA and hRAD51 are equivalent, it is tempting to speculate that there is significantly less hRAD51 protein in the hRAD51·DNA_low form than RecA protein in the RecA·ssDNA complex.

There does not appear to be a clear correlation between IAB hRAD51·ssDNA complexes and GS hRAD51·ssDNA forms. In general, it appears that the IAB hRAD51·ssDNA complexes are representative of both the GS hRAD51·DNA_low and hRAD51·DNA_high forms. The cumulative GS binding of hRAD51·ssDNA in both the low and high mobility forms appears to be equivalent to the results obtained with IAB. However, we have found that only the hRAD51·DNA_low form appears capable of adenosine nucleotide processing. For example, the subsequent addition of ATPS dissociates the hRAD51·DNA_low form but not the hRAD51·DNA_high form (see Fig. 5). In addition, ADP  ATP exchange can be measured at low ADP concentrations (20 µM) when the hRAD51·DNA_low form appears to be exclusive (10). However, we were unable to detect ADP  ATP exchange by hRAD51 at high ADP concentrations (200 µM) when it is largely in the hRAD51·DNA_high form (10). These results are consistent with the notion that the hRAD51·DNA_low form is active for both adenosine nucleotide processing and ssDNA binding. It is likely that the hRAD51·DNA_high form represents an aggregate structure. This conclusion is based on its inability to enter the gel matrix and our finding that it can be pelleted by centrifugation. Since this hRAD51·DNA_high form appears irreversible and correlates with a secondary nonsaturable mode of ADP binding (see Ref. 10), it is likely to be a nonspecific aggregate that is only important for in vitro studies where incubation times exceed 1 h or where the formation of significant levels of ADP occur. Such conditions may be routinely used in hRAD51-mediated strand exchange studies.

RecA efficiently discriminates ADP, ATP, and ATPS (1). This discrimination is manifest in cooperative ATP hydrolysis and recombinational strand exchange. In contrast, hRAD51 is unable to adequately differentiate ADP, ATP, and ATPS. IAB and GS studies indicate that hRAD51 forms similar ssDNA complexes in the presence of all three of these adenosine nucleotides (below 150 µM). In addition, the hRAD51 binding constant (K_D or K_app1) for ATP/ATPS and ADP is equivalent. Under similar circumstances, RecA is capable of discriminating between ADP and ATP/ATPS as well as between ATP and ATPS. The inability to efficiently discriminate between adenosine nucleotides provides a foundation for the idea that hRAD51 function(s) requires additional protein factors to duplicate RecA function(s). Potential candidates for hRAD51 co-factor(s) are one or all of the known mitotic or meiotic human RecA homologs: hRAD51B, hRAD51C, hRAD51D, XRCC2, XRCC3, and hDMC1 (47-49).

	ACKNOWLEDGEMENTS

We thank Hans-Jürg Alder and the Kimmel Nucleic Acids Facility for oligonucleotide synthesis and sequencing, and we thank Chris Schmutte, Samir Acharya, Scott Gradia, and Kristine Yoder for helpful discussions and careful review of the manuscript.

	FOOTNOTES

^* This work was supported by National Research Service Award Grant 5-T32-CA09678 (to G. T.) and National Institutes of Health Grant CA56542 (to R. F.).

To whom correspondence and reprint requests should be addressed: Kimmel Cancer Center, BLSB933, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 213-503-1346; Fax: 215-923-1098; E-mail: rfishel@ hendrix.jci.tju.edu.

Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M109917200

	ABBREVIATIONS

The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ATPase, ATP hydrolysis activity; hRAD51, human RAD51 protein; ATPS, adenosine 5'-O-(thiotriphosphate); IAB, IAsys biosensor; GS, gel shift.

	REFERENCES

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