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Genes to Cells (2008) 13, 471-481. doi:10.1111/j.1365-2443.2008.01180.x
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Inhibition of filament formation of human Rad51 protein by a small peptide derived from the BRC-motif of the BRCA2 protein

 Julian Nomme1, Yoshimasa Takizawa2, Susan F. Martinez1, Axelle Renodon-Cornière1, Fabrice Fleury1, Pierre Weigel1, Ken-ichi Yamamoto3, Hitoshi Kurumizaka2 and Masayuki Takahashi1,*

1 UMR 6204, Centre National de la Recherche Scientifique, Université de Nantes, 44322 Nantes cedex 3, France
2 Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
3 Research Institute of Cancer, Kanazawa University, 13-1 Takara-machi, Kanazawa-shi 920-0934, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Human Rad51 is a key element of recombinational DNA repair and is related to the resistance of cancer cells to chemo- and radiotherapies. The protein is thus a potential target of anti-cancer treatment. The crystallographic analysis shows that the BRC-motif of the BRCA2 tumor suppressor is in contact with the subunit–subunit interface of Rad51 and could thus prevent filament formation of Rad51. However, biochemical analysis indicates that a BRC-motif peptide of 69 amino acids preferentially binds to the N-terminal part of Rad51. We show experimentally that a short peptide of 28 amino acids derived from the BRC4 motif binds to the subunit–subunit interface and dissociates its filament, both in the presence and absence of DNA, certainly by binding to dissociated monomers. The inhibition is efficient and specific for Rad51: the peptide does not even interact with Rad51 homologs or prevent their interaction with DNA. Neither the N-terminal nor the C-terminal half of the peptide interacts with human Rad51, indicating that both parts are involved in the interaction, as expected from the crystal structure. These results suggest the possibility of developing inhibitors of human Rad51 based on this peptide.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The Rad51 protein is a key element of homologous recombination and is thus involved in DNA repair and DNA segregation (Shinohara et al. 1992; Sung 1994; Haaf et al. 1995; Baumann et al. 1996; Maeshima et al. 1996; Gupta et al. 1997; Sonoda et al. 1998; Vispe et al. 1998). The protein plays a dual role in cancer development: its DNA repair capacity prevents cancer formation but, once a cancer has formed, it protects cancer cells against anti-cancer treatments, such as radio- and chemotherapies (Yanagisawa et al. 1998; Christodoulopoulos et al. 1999; Henning & Sturzbecher 2003), which damage DNA in order to kill cancer cells. Furthermore, high recombination activity without proper regulation could promote chromosomal translocation and loss of heterozygosity, thus generating more malignant cancer cells (Christodoulopoulos et al. 1999). Rad51 is also involved in cell proliferation because of its participation in DNA segregation. Rad51 is frequently over-expressed in tumor cells (Yanagisawa et al. 1998; Maacke et al. 2000; Henning & Sturzbecher 2003) and certainly contributes to their proliferation. There are some correlations between the cellular amount of Rad51 and the advancement of cancer as well as between this amount and the resistance of cells to chemotherapy (Yanagisawa et al. 1998; Christodoulopoulos et al. 1999; Maacke et al. 2000; Henning & Sturzbecher 2003). Inhibiting its production by antisense and RNAi strategies slows down tumor development and increases the efficiency of radiotherapy (Ohnishi et al. 1998; Collis et al. 2001; Ito et al. 2005). Rad51 is thus a potential target of anti-cancer treatment.

To prepare inhibitors of human Rad51 (HsRad51), we examined the inhibitory effect of short peptides derived from the BRC-motif of the BRCA2 protein. BRCA2 is a tumor suppressor involved in the regulation of DNA repair (Wooster et al. 1995; Sharan et al. 1997; Boulton 2006). It interacts with HsRad51, transports it into the nucleus and thus activates homologous recombination (Sharan et al. 1997; Wong et al. 1997; Chen et al. 1999; Yuan et al. 1999). Its interaction with HsRad51 occurs via conserved regions called the BRC-motif, which is repeated 8 times in BRCA2 (Bork et al. 1996; Bignell et al. 1997; Wong et al. 1997). Each motif is constituted of about 70 amino acids with a conserved sequence of 24 amino acids (Bork et al. 1996; Bignell et al. 1997; Wong et al. 1997). When expressed in cells, some BRC-motif fragments inhibit the recombinase activity of HsRad51 (Sharan et al. 1997; Chen et al. 1999; Yuan et al. 1999). The structure of the BRC-motif–HsRad51 complex has been determined by crystallographic analysis of a fusion protein in which the central part of HsRad51 is fused to one of the BRC-motifs (BRC4) with a small linker (Pellegrini et al. 2002). The analysis has shown that the BRC-motif is in contact with the hydrophobic pocket around Tyr191 in HsRad51. This part is considered to be one of the subunit–subunit interfaces (Pellegrini et al. 2002; Selmane et al. 2004). The BRC-motif could thus prevent subunit–subunit contact, which is required for the formation of the Rad51–DNA complex filament, the first step of the strand exchange reaction in homologous recombination. In fact, inhibition of the HsRad51–DNA complex formation was observed in vitro using a peptide of about 70 amino acids (Davies et al. 2001).

However, it was reported that a peptide of 69 amino acids derived from the BRC3 motif binds primarily to the N-terminal domain of HsRad51 instead of the subunit–subunit interface (Galkin et al. 2005). The dissociation of the HsRad51–DNA complex filament occurred only at higher concentrations of the peptide. This discrepancy with the crystallographic analysis could be due to the absence of the N-terminal domain of HsRad51 or the use of a shorter peptide (33 instead of 69 amino acids) in the crystallographic analysis, or to the inaccessibility of the subunit–subunit interface in the complex with DNA. To discriminate these options and examine the potential of using a short BRC-motif peptide to inhibit the filament formation of HsRad51, we have investigated, in this work, the effect of shorter peptides on the filament formation of HsRad51 in the presence and absence of DNA. The inhibition in the absence of DNA was examined by gel filtration chromatography and chemical cross-linking of HsRad51. The inhibition of filament formation in the presence of DNA was studied by measuring the fluorescence change of the poly(dA) analog, poly(deoxy-1,N6-ethenoadenylic acid) (poly(d{varepsilon}A)), and by gel shift analysis. The fluorescence intensity of poly(d{varepsilon}A) greatly increases upon binding Rad51 (Maeshima et al. 1998), and can thus be used to detect the formation and dissociation of the complex in solution and in real time, enabling a kinetic analysis. The study was performed both in the presence and absence of ATP, which is required for the strand exchange activity of Rad51.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Design of inhibitory peptides

Visual inspection of the crystal structure of the BRC4–HsRad51 complex (Pellegrini et al. 2002) indicates that the residues outside of 1521 and 1549 of BRCA2 are not involved in the interaction. We therefore prepared a peptide of 28 amino acids spanning residues 1521–1548 of BRCA2 (BRC4-28). Our computation of the interaction energy of each residue of the BRC4 motif with HsRad51 in the crystal structure (Pellegrini et al. 2002) confirmed our conclusion (Fig. 1). The binding energy was computed using the DOCKING option (InsightII, Accelrys) (Sali & Blundell 1993), which takes into account the electrostatic and Van der Waals non-bonded energies. It showed that two parts of the BRC4 motif, around residue 1524 and residue 1546, are in strong contact with HsRad51. Since the peptide can be structurally divided into two parts (β-turn and {alpha}-helix) and each part interacts separately with HsRad51 in the crystal (Fig. 1) (Pellegrini et al. 2002), we tested the inhibitory effect of each peptide containing only one of the contact parts: one from 1521 to 1533 (BRC4-13) and the other from 1534 to 1548 (BRC4-15).


Figure 1
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  		Figure 1  Structure and contacts of the BRC4 motif of the BRCA2 protein with HsRad51. The amino acid sequence of the BRC4 motif is noted with its secondary structure in the complex with HsRad51 (Pellegrini et al. 2002). The residues that are in stronger contact with HsRad51 are indicated in a darker color. The 3D structure of the peptide in the complex is also represented.

 
Structure of peptides and interaction with Rad51

We examined the structure of the peptides by circular dichroism (CD) measurements. All of them exhibited a CD signal corresponding to a random coil structure (Fig. 2). The peptides could not form any stable secondary structure in aqueous solution whereas the BRC4 motif forms short anti-parallel β-sheets at the N-terminal part and a short {alpha}-helix at the C-terminal part in the complex with HsRad51 (Pellegrini et al. 2002) (Fig. 1). When 1 mM sodium dodecyl sulfate (SDS) was added to generate a hydrophobic environment, the CD signal of the BRC4-28 peptide was largely modified in a way indicating the formation of an {alpha}-helix (Fig. 2A). The peptide certainly has a strong tendency to form an {alpha}-helix because SDS usually promotes a β-sheet structure at such a low concentration (Waterhouse & Johnson 1994). Addition of 1 mM SDS also modified the CD signal of the C-terminal half peptide (BRC4-15) (Fig. 2C) but did not greatly affect that of the N-terminal half (BRC4-13) (Fig. 2B), suggesting that the {alpha}-helix is formed at the C-terminal part of the BRC4-28 peptide, as expected from the crystal structure of the BRC-motif–HsRad51 complex (Fig. 1) (Pellegrini et al. 2002). These results also suggest that separation of the BRC4-28 peptide into two parts does not prevent the folding of the C-terminal half.


Figure 2
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  		Figure 2  CD spectra of the BRC4-motif peptides. CD spectra of the BRC4-28 (panel A), its N-terminal half (BRC4-13) (panel B) and C-terminal half (BRC4-15) (panel C) peptides (0.05 mg/mL) were measured at 25 °C in the presence (continuous line) and absence (broken line) of 1 mM SDS to analyze their secondary structure.

 
We then examined peptide interactions with HsRad51 by monitoring changes in the fluorescence anisotropy of fluorescein covalently attached to the peptides. Fluorescence anisotropy is related to the motion of a fluorophore and is greater when this is stiffly attached to larger molecules (Lakowicz 1999). The anisotropy is therefore expected to increase upon formation of a complex with a large molecule like HsRad51. The experiments were carried out both in the presence and absence of ATP. ATP is required for the strand exchange activity of Rad51 (Chi et al. 2006) and affects the filament structure and the DNA binding of Rad51 (Ellouze et al. 1997; Yu et al. 2001; Tombline et al. 2002). ATP is hydrolyzed only slowly by Rad51.

Stepwise addition of HsRad51 to 0.2 µM BRC4-28 peptide increased the fluorescence anisotropy (Fig. 3). The change was almost saturated with 0.3 µM of HsRad51 in the presence of ATP. The half-effect was achieved at around 0.1 and 0.3 µM in the presence and absence of ATP, respectively. Thus, the peptide binds to HsRad51 more efficiently in the presence of ATP than in its absence.


Figure 3
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  		Figure 3  Specific binding of BRC4-motif peptides to HsRad51. In panel A, binding of the BRC4-28 peptide to HsRad51 in the presence (open diamonds) and absence (closed diamonds) of 1 mM ATP was monitored by the change in the fluorescence anisotropy of fluorescein-labeled peptide (0.2 µM) upon stepwise addition of HsRad51. The results are presented as a function of HsRad51 concentration. No significant change was observed upon addition of RecA (crosses) or Dmc1 (dashes) indicating an absence of binding. In panel B, binding of the half peptides, BRC4-13 (triangles) and BRC4-15 (squares), to HsRad51 was tested in the presence (open symbols) and absence (closed symbols) of 1 mM ATP by measuring the fluorescence anisotropy of the labeled peptides (0.2 µM) after addition of HsRad51. The results with BRC4-28 (diamonds) are also shown.

 
As a negative control, we tested the binding of a peptide whose amino acid composition was the same as that of BRC4-28 peptide but whose sequence was not related. No significant increase in fluorescence anisotropy was observed upon addition of HsRad51 up to 0.65 µM (not shown), indicating no interaction. The specific amino acid sequence of BRC4-28 is thus required for the interaction with HsRad51. The interaction was also specific for HsRad51: adding its homologs, RecA or Dmc1, did not significantly affect the anisotropy (Fig. 3A), certainly reflecting the absence of interaction of BRC4-28 with these proteins.

We also tested whether the fragments of BRC4-28 interact with HsRad51. The addition of HsRad51 to the N-terminal or C-terminal half peptide in the presence or absence of ATP did not affect the anisotropy (Fig. 3B), indicating no significant binding. Clearly, when separated into two fragments, the peptide could not interact with HsRad51. Thus, both parts of BRC4-28 are required for efficient interaction, suggesting that both the N- and C-terminal parts are involved in this interaction, as observed by crystallographic analysis of the complex (Pellegrini et al. 2002). We note that the anisotropy values of the N- and C-terminal half peptides were less than that of the 28-amino acid peptide (in the absence of protein), reflecting their smaller size.

Inhibition of self-assembly of Rad51

Like RecA protein, HsRad51 forms oligomers even in the absence of DNA (Ellouze et al. 1997; Yoshioka et al. 2003; Matsuo et al. 2006). HsRad51 was eluted almost at void volume from a Superdex 200 molecular sieve chromatography column, both in the presence and absence of ATP (Fig. 4A,C), indicating apparent molecular mass of more than 200 kDa. Since the molecular mass of the HsRad51 monomer is 38 kDa, the results confirm auto-association of HsRad51. However, after incubation with the BRC4-28 peptide, a part of HsRad51 was eluted at a position corresponding to a molecular mass of about 40 kDa, both in the presence and absence of ATP (Fig. 4B,D and E). Therefore, HsRad51 was dissociated into monomers in the presence of the peptide. The dissociation was not complete, no doubt because the high protein concentration (10 µM) favors filament formation. We also verified this dissociation in the presence of ATP by analyzing the chemical cross-linking of HsRad51. Electrophoretic analysis of HsRad51, which was treated with a cross-linking reagent, revealed the formation of cross-linked dimers, confirming the auto-association of HsRad51. With increasing concentration of BRC4-28 peptide, the amount of cross-linked dimer decreased and cross-linked products of HsRad51-BRC4-28 appeared (Fig. 4F), supporting the dissociation of HsRad51 filament by BRC4-28.


Figure 4
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  		Figure 4  Inhibition of the filament formation of HsRad51 by the BRC4-28 peptide. 10 µM HsRad51 was loaded onto and eluted from a Superdex 200 gel filtration column with (B and D) and without (A and C) 40 µM BRC4-28 peptide in the presence (C and D) and absence (A and B) of ATP to examine the effect of peptide on the polymerization state of HsRad51. The quantity of HsRad51 in every two fractions was verified by SDS gel electrophoresis analysis with silver staining. The molecular mass corresponding to the elution peak of HsRad51-BRC4-28 (indicated by an arrow) was estimated from the relation between the elution position (Kav = (Ve – Vo)/(Vt – Vo) where Ve is the elution volume for the protein, Vo is the column void volume and Vt is the total bed volume) and the molecular mass of standards (Ferritin, Aldolase, Conalbumin, Ovalbumin, Carbonic Anhydrase, Ribonuclease, and Aprotinin) (panel E). In panel F, the inhibition of filament formation by BRC4-28 in the presence of ATP (1 mM) was examined by chemical cross-linking of HsRad51 (2.5 µM) as described in Experimental Procedures. The cross-linked products were separated by electrophoresis on SDS polyacrylamide gel and detected by Western Blotting using anti-Rad51 antibody. The concentrations of BRC4-28 are noted at the top of each column. The molecular mass indicators are shown in the first column.

 
Inhibition of DNA binding

Inhibition of the filament formation of the HsRad51–DNA complex was then monitored by the fluorescence change of poly(d{varepsilon}A), a fluorescent analog of poly(dA). Its fluorescence greatly increases in intensity upon Rad51 binding (Maeshima et al. 1998; Kim et al. 2002) so can be used to monitor the formation and dissociation of the Rad51–DNA complex in real time. Binding of HsRad51 increased the fluorescence of poly(d{varepsilon}A) about fourfold in intensity in the absence of ATP and sevenfold in its presence, as reported previously for Xenopus Rad51 (Maeshima et al. 1998). Adding the BRC4-28 peptide to the HsRad51–poly(d{varepsilon}A) complex almost annulled this fluorescence increase, indicating almost complete dissociation of the HsRad51–poly(d{varepsilon}A) complex (Fig. 5). Half-dissociation of the complex formed with 1.5 µM HsRad51 and 3 µM poly(d{varepsilon}A) was achieved with 2.2 µM peptide in the presence of ATP and 7 µM in its absence (Fig. 5). Here again we observed a more efficient effect of the peptide in the presence of ATP. The dissociation of the HsRad51–oligo(dA) complex by this peptide was also verified by a gel shift experiment: the shifted band disappeared upon addition of the peptide with the reappearance of the band corresponding to free DNA (data not shown).


Figure 5
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  		Figure 5  Dissociation of the HsRad51–DNA complex by the BRC4 peptides. The dissociation of the HsRad51-poly(d{varepsilon}A) complex by BRC4-28 was analyzed in the presence (open diamonds) and absence (closed diamonds) of 1 mM ATP by measuring the fluorescence of poly(d{varepsilon}A) after each stepwise addition of the peptides. The inhibitory effect of modified BRC4-28, in which the threonine residue at position 1526 was substituted with alanine, was also examined in the presence (open circles) and absence (closed circles) of 1 mM ATP. The concentration of HsRad51 was 1.5 µM and that of poly(d{varepsilon}A) 3 µM. (1.0 : 100% HsRad51–DNA complex).

 
To verify the specificity of the peptide for the inhibition of HsRad51, we tested the inhibitory activity of the peptide in which threonine at position 1526 was substituted with alanine. This threonine residue contributes to the interaction with HsRad51 according to the crystallographic analysis (Pellegrini et al. 2002). Furthermore, the corresponding substitution in the BRC3-motif peptide is reported to abolish the inhibitory effect (Davies et al. 2001). The substitution also affected the inhibitory effect of the BRC4-28 peptide (Fig. 5): the modified peptide could not inhibit the filament formation of the HsRad51–poly(d{varepsilon}A) complex in the absence of ATP. Even in the presence of ATP, the modified peptide was much less efficient than the original BRC4-28 peptide (Fig. 5). These results clearly show the importance of a specific amino acid sequence for the inhibitory activity as expected and suggest that the inhibition mechanisms of BRC3 and BRC4 peptides are similar.

Mechanism of inhibition of DNA binding

We examined whether the inhibitory effect of the BRC4-28 peptide is really due to direct interaction of the peptide with HsRad51 or via interaction of the peptide with DNA. Addition of peptide alone did not affect the fluorescence of poly(d{varepsilon}A), indicating no direct interaction between the peptide and poly(d{varepsilon}A). Furthermore, the fluorescence increase of poly(d{varepsilon}A) upon complex formation with RecA or Dmc1, to which the BRC4-28 peptide could not bind, was not annulled by adding the BRC4-28 peptide (data not shown). BRC4-28 did not dissociate the RecA-DNA or Dmc1-DNA complex. This observation is in accord with the idea that the peptide dissociates the complex via interaction with the protein. Further supporting this idea, the half-size peptides (BRC4-13 and BRC4-15), which do not bind to HsRad51, did not significantly dissociate the HsRad51–poly(d{varepsilon}A) complex (data not shown).

There are two possible mechanisms for the dissociation of the HsRad51-DNA complex: (i) the peptide binds to the HsRad51-DNA filament and destabilizes the complex or (ii) the peptide binds to HsRad51 molecules dissociated from the DNA, and prevents their re-association with DNA. In the first case, the destabilization step is usually slow and thus the dissociation kinetics are independent of inhibitor concentration while for the second model, the kinetics depend upon inhibitor concentration. We therefore examined the effect of inhibitor concentration on the dissociation kinetics. The kinetics were followed by monitoring the fluorescence change of poly(d{varepsilon}A). The dissociation was rather slow, especially in the absence of ATP, taking several minutes (Fig. 6). The dissociation rate depended upon the concentration of BRC4-28 peptide in a way that suggests that the association of peptide is the limiting step (Fig. 6A). Thus, these results reject the reaction mechanism in which the BRC4-28 peptide binds to the HsRad51–DNA complex first and then slowly modifies the complex conformation to promote dissociation. Since the association of BRC4-28 peptide to free HsRad51 without DNA is fast and occurred within the mixing time (< 1 min), the slow dissociation of the HsRad51–DNA complex suggests that the site for the binding of BRC4-28 peptide is not well accessible in the HsRad51–DNA complex. The observation is in good accord with the idea that the peptide binds to the subunit–subunit interface. BRC4-28 probably binds only to the HsRad51 molecules dissociated from the DNA. This conclusion is further supported by the observation that the BRC4-28 peptide could not efficiently dissociate the adenosine 5'-(β,{gamma}-imido) triphosphate (AMP–PNP)–HsRad51–poly(d{varepsilon}A) or adenosine 5'-O-3-thiophosphate (ATP{gamma}S)–HsRad51–poly(d{varepsilon}A) complexes (Fig. 6B), from which HsRad51 dissociates very slowly (Mine et al. 2007). This is not due to the inefficacity of BRC4-28 peptide for the inhibition in the presence of these nucleotide analogs because no formation of HsRad51–poly(d{varepsilon}A) complex was observed even in their presence when HsRad51 was incubated with the peptide before mixing with the DNA. Once bonded to HsRad51, the peptide efficiently prevents the association of HsRad51 to the DNA.


Figure 6
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  		Figure 6  Dissociation kinetics of the HsRad51-poly(d{varepsilon}A) complex by the BRC4-28 peptide: effects of peptide concentration and nucleotides. The BRC4-28 peptide-induced dissociation kinetics of HsRad51-poly(d{varepsilon}A) complex were followed by changes in the fluorescence of poly(d{varepsilon}A) as follows: the HsRad51-poly(d{varepsilon}A) complex was first formed (phase 2) by addition of 1.5 µM HsRad51 to preincubated 3 µM (in bases) poly(d{varepsilon}A) (phase 1) at time 7 min, and then BRC4-28 was added at time 40 min and the dissociation was observed (phase 3). In panel A, the effect of BRC4-28 concentration (40 µM: diamonds and 10 µM: dashes) on the dissociation was examined in the absence of ATP. In panel B, the effect of nucleotides was examined by performing the same experiments in the presence of 1 mM AMP-PNP (crosses), ATP{gamma}S (open triangles) or ATP (open diamonds) and using 40 µM BRC4-28 peptide.

 
This inhibition mechanism is in accord with the idea that the BRC4-28 peptide binds to the subunit–subunit interface of HsRad51, which should not be accessible in the HsRad51–DNA complex filament.

Inhibition of DNA strand exchange

Finally, we tested the inhibitory effect of the BRC4-28 peptide on the DNA strand exchange activity of HsRad51. The strand exchange reaction between a short single-stranded oligonucleotide and a double-stranded oligonucleotide of the same sequence was performed in the presence of various concentrations of the BRC4-28 peptide. As expected, the strand exchange reaction was greatly reduced by the peptide (Fig. 7).


Figure 7
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  		Figure 7  Inhibition of the DNA strand exchange reaction by the BRC4-28 peptide. The HsRad51-promoted (0.5 µM) strand exchange reaction between single-stranded and double-stranded oligonucleotides in the presence of indicated concentrations of BRC4-28 peptide was analyzed using polyacrylamide gel electrophoresis as described in Experimental Procedures.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Our study clearly shows that the small peptide of 28 amino acids derived from the BRC-motif of the BRCA2 protein can efficiently and selectively interact with HsRad51 and inhibit the formation of the HsRad51–DNA complex filament and thus the strand exchange reaction. Our results in solution with entire HsRad51 are in good agreement with the crystallographic analysis of the HsRad51–BRC-motif peptide complex (Pellegrini et al. 2002) despite the absence of the N-terminal part of HsRad51 in the crystal. More efficient inhibitors could, therefore, be developed from this peptide using the crystallographic analysis.

Our results, including the kinetic analysis, indicate that the short peptide binds to the subunit–subunit interface of HsRad51 as observed in the crystal, although we cannot exclude the possibility that the peptide also binds to the N-terminal domain of HsRad51. The reason for the preferential binding of longer BRC-motif peptides (69 amino acids) to the N-terminal domain of HsRad51 in the complex with DNA (Galkin et al. 2005) is probably that the subunit–subunit interface of HsRad51 is not accessible to the peptide when bound to DNA. It is also possible that some parts of the peptide outside the 28 amino acids of BRC4-28 facilitate binding to the N-terminal domain or prevent binding to the interface. Our observation does not reject the possibility of an interaction of the entire BRCA2 with the N-terminal domain of HsRad51, but it clearly shows that the short BRC-motif peptide of 28 amino acids binds to the subunit–subunit interface and inhibits the filament formation of HsRad51.

The observation that neither the N-terminal nor the C-terminal half of BRC4-28 peptide can efficiently interact alone with Rad51 supports the crystal structure where both parts are in interaction with HsRad51. Our results obtained in solution and using entire HsRad51 correspond well with the crystallographic analysis of the BRC-HsRad51 fusion protein in which only the central part of HsRad51 is present. Furthermore, we observed that the peptide tends to form an {alpha}-helix at the C-terminal part in a non-polar environment, that is, in contact with the protein, as observed in the crystal of the complex with HsRad51 (Pellegrini et al. 2002). The peptide should interact with entire HsRad51 in a similar way to with truncated HsRad51 in the crystal. This crystal structure can certainly be exploited to design more efficient inhibitors.

The observation that the substitution of threonine at position 1526 with alanine decreases the inhibitory capacity further supports the validity of the crystal structure, which shows the importance of this threonine residue for the interaction with HsRad51 (Fig. 1). This observation also suggests that the BRC3 motif inhibits HsRad51 in a similar way to BRC4-28 because the substitution of the corresponding threonine in the BRC3-motif peptide abolishes its inhibitory activity (Davies et al. 2001). However, the effect of substitution appears more severe in BRC3 than in BRC4. It almost completely abolishes the inhibitory activity of the BRC3-motif peptide while it leaves some residual activity for BRC4-28. The binding energy of the other parts of BRC4-28, probably the C-terminal half, is greater than that of the BRC3 motif, and allows some inhibitory activity. Our preliminary experiments show that the BRC4-motif peptide, missing three amino acids at the N-terminal part of BRC4-28, can still inhibit HsRad51 (data not shown) while the corresponding BRC3-motif peptide does not exhibit any inhibitory effect (Davies et al. 2001). Thus, the BRC4 motif appears to be the best one for HsRad51 binding.

BRC4-28 is specific for Rad51, indicating the potential use of this peptide for anti-cancer treatment targeting the Rad51 protein. Even its prokaryote homologue, RecA, or its homologue of meiotic recombinase, Dmc1, cannot interact with the peptide. The polymerization motif of HsRad51, which is involved in the subunit–subunit contact of HsRad51 (Pellegrini et al. 2002), does not show very high sequence identity with that of Escherichia coli RecA. The amino acid sequence of HsRad51 is GFTTATE while that of RecA is SIMRLGE (Pellegrini et al. 2002). This may explain the specificity of the peptide towards Rad51. In the case of Dmc1, the sequence of the polymerization motif is rather similar (GFLTAFE) but not identical. Furthermore, Dmc1 forms a stable octameric ring even in the absence of DNA (Masson et al. 1999; Kinebuchi et al. 2004) so the part may not be available to the peptide. In contrast, the association to and dissociation from the filament of HsRad51 without DNA is rather fast (Yoshioka et al. 2003) thus the interface could be accessible to the BRC-motif peptide.

The more efficient binding of BRC4-28 to HsRad51 in the presence than in the absence of ATP indicates some structural changes in the subunit–subunit contact sites of HsRad51 upon binding of ATP. These changes could affect the subunit–subunit contact in some way and could be the origin of the ATP-induced elongation of the Rad51 filament (Ellouze et al. 1997). The ATP-binding site of Rad51 is rather close to the subunit–subunit interface according to the crystal structure of yeast Rad51 filament (Conway et al. 2004) and thus ATP binding could affect the structure of the subunit–subunit interface. The peptide could also be used to investigate the subunit–subunit interaction of HsRad51.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Preparation of proteins

The N-terminal hexahistidine-tagged HsRad51 was expressed in the JM109(DE3) strain of E. coli that also carries an expression vector for the minor tRNAs (Codon(+)RIL®, Novagen, Darmstadt, Germany). The protein was purified on nickel-nitrilotriacetic acid (Ni-NTA) agarose (Invitrogen, Carlsbad, CA). The hexahistidine tag was then removed from the Rad51 portion with thrombin (Amersham Biosciences, Uppsala, Sweden) and the protein was further purified by chromatography on a MonoQ column (Healthcare Biosciences. Little Chalfont, UK). Protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin (Pierce, Rockford, IL) as a standard protein. Human Dmc1 and E. coli RecA were prepared as described elsewhere (Kurumizaka et al. 1996; Kinebuchi et al. 2004).

Peptides

The peptides were prepared by NeoSystem (France). Their amino acid composition and purity (> 95%) were verified by mass spectroscopy by the manufacturer. BRC4-28 is composed of 28 amino acids spanning the sequence of the BRC4 motif (1521–1548 of BRCA2). BRC4-13 is 13 amino acids of the N-terminal part (1521–1533) of BRC4-28, and BRC4-15 is 15 amino acids of the C-terminal part (1534–1548) of BRC4-28 (Fig. 1). A peptide of 28 amino acids whose amino acid composition was identical to BRC4-28 but whose sequence was shuffled was also prepared (GGLKDLKDFEKLISALKHVFKEATKNSV). The BRC4-28 peptide in which threonine at position 1526 was substituted with alanine was prepared by Professor Kazuyasu Sakaguchi (Hokkaido University, Sapporo, Japan). Fluorescein-labeled peptides were also prepared by NeoSystem. Fluorescein was attached to the N-terminal part.

Oligonucleotides

Poly(d{varepsilon}A) was prepared by chemical modification of poly(dA) (Pharmacia) as described in (Chabbert et al. 1991). IRD-labeled oligo(dA), 32 bases in length, and the following oligonucleotides were obtained from MWG and used without further purification:

59-mer 5'-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GGC TTA GAG CTT AAT TGC TGA ATC TGG TG-3'
32-mer 5'-CCA TCC GCA AAA ATG ACC TCT TAT CAA AAG GA-3'
32-mer * 5'-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GG-3'.

The 5' end of the last oligonucleotide was labeled with IRD-800.

Other chemicals

ATP, AMP–PNP, and ATP{gamma}S were obtained from Sigma (St Louis, MO).

Fluorescence measurements

Fluorescence was measured in an FP-6500 spectrofluorometer (Jasco, Japan) using a 0.2 x 1 cm mini cell (Hellma, Nurnberg, Germany). The fluorescence of poly(d{varepsilon}A) was observed at 410 nm (bandwidth: 5 nm) with excitation at 320 nm (bandwidth: 3 nm). Fluorescence anisotropy of the fluorescein-labeled peptides was measured at 520 nm (bandwidth: 5 nm; response time: 0.5 s) upon excitation at 450 nm (bandwidth: 5 nm). Anisotropy (r) was computed using the formula, r = (IVV – G x IVH)/(IVV + 2G x IVH) where I is the fluorescence intensity, with the first index noting the orientation of the exciting polarizer and the second that of the analyzer (V: vertical, H: horizontal). G is a correction factor defined as G = (IHV/IHH). Most experiments were performed at 25 °C in 20 mM (in phosphate) sodium phosphate buffer, pH 7.4 containing 50 mM NaCl and 1 mM MgCl2. The buffer was filtered on a nitrocellulose filter (pore size: 0.2 µm) (Pall Corporation, East Hills, NY) and degassed.

CD measurements

CD spectra were measured with step mode (bandwidth: 2 nm; data interval: 0.1 nm; response time: 0.125 s) in a J-810 CD spectrometer (Jasco) equipped with a Peltier effect temperature controller. The spectra were averaged over three scans to increase the signal to noise ratio. A 1 x 0.2 cm mini quartz cell with four windows (Hellma) was used. The path length was usually 0.2 cm.

Gel filtration

HsRad51 (10 µM) was loaded on a Superdex 200 10/300 (Healthcare Biosciences) gel filtration column, and eluted with a buffer containing 20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 2 mM 2-mercaptoethanol, and 10% glycerol, and the flow rate was 0.5 mL/min. The elution pattern was monitored by UV absorption at 280 nm. The samples were incubated for 1 h at 37 °C.

Chemical cross-linking of HsRad51

The reaction was performed at room temperature. 2.5 µM HsRad51 was incubated with the indicated concentrations of BRC4-28 peptide for 10 min in a buffer containing 20 mM sodium phosphate, 50 mM NaCl, 1 mM MgCl2, and 1 mM ATP. The cross-linking reactions were initiated by the addition of disuccinimidyl suberate (50 µM) and allowed to proceed for 30 min. Reactions were quenched by the addition of Tris–HCl (pH 7.5, final concentration: 50 mM) followed by subsequent incubations for 15 min. The products were then separated by 12% SDS polyacrylamide gel electrophoresis and detected by Western blot with mouse monoclonal anti-Rad51 antibody (NeoMarkers) and anti-mouse Alexa-Fluor 700-conjugated secondary antibody (Molecular Probes, Eugene, OR). The cross-linked dimer was quantified by an Odyssey Infrared Imaging System scanner (Li-Cor Biosciences).

Strand exchange assay

The DNA strand exchange reaction was performed as described in (Takizawa et al. 2004) with minor modifications. HsRad51 protein was incubated with the indicated amounts of BRC4-28 peptide and the 59-mer single-stranded DNA (100 nM in fragment) in a buffer containing 20 mM Tris–HCl (pH 8.0), 1 mM ATP, 1 mM DTT, 100 µg/mL BSA, 20 mM MgCl2, 2 mM creatine phosphate, 75 µg/mL creatine kinase, and 2% glycerol, at 37 °C for 15 min. The reaction was initiated by adding the IRD-labeled 32-mer dsDNA (10 nM in fragment), which shared sequence homology with the 59-mer ssDNA. After 1 h of incubation at 37 °C, the reactions were stopped by the addition of 0.7% SDS, and 0.7 mg/mL proteinase K (Roche Molecular Biochemicals, Indianapolis, IN). The reaction mixtures were further incubated for 10 min at 37 °C. After addition of loading dye, the reaction products were subjected to electrophoresis on 15% polyacrylamide gel. The products were visualized by detection of IRD-800. Three independent measurements were performed and averaged. 0.5 µM HsRad51 was usually used because the formation of strand exchange product was proportional to the HsRad51 concentration from 0.5 to 1.5 µM with a reaction time of up to 1 h with this protein concentration. In contrast, we observed inhibition of the strand exchange reaction by high concentrations of HsRad51 (more than 2 µM), no doubt because 100 nM oligonucleotides of the 59-base is saturated with 2 µM HsRad51 and the excess protein binds to the double-stranded DNA preventing the reaction (Mine et al. 2007).


   Acknowledgements
 
We thank Professor Vinh Tran and Dr. Sébastien Conilleau (Nantes University, France) for discussion, and Professor Kazuyasu Sakaguchi (Hokkaido University, Japan) for the preparation of peptide. This work was supported by grants from the Association pour la Recherche sur le Cancer (No. 3862 and No. 7670), the Ministry of Education and Research of the French Government (Action Concertée), the Japanese Society for the Promotion of Science (JSPS) and the Ministry of Education, Sports, Culture, Science, and Technology of Japan. JN is a recipient of a PhD fellowship from the Ligue contre Cancer Comité Loire Atlantique and SM from the Ambassador of France-CONICYT (Chile). MT thanks Jasco International (Hachioji, Japan) for kindly providing a spectrofluorometer (FP6500, Jasco) for some measurements.


   Footnotes
 
Communicated by: Moshe Yaniv

* Correspondence: Email: masayuki.takahashi{at}univ-nantes.fr


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 26 October 2007
Accepted: 4 February 2008




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