GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2008) 13, 91-103. doi:10.1111/j.1365-2443.2007.01143.x
© 2008 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishida, T.
Right arrow Articles by Kurumizaka, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Ishida, T.
Right arrow Articles by Kurumizaka, H.

The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity

 Takako Ishida1, Yoshimasa Takizawa1, Isao Sakane2 and Hitoshi Kurumizaka1,2,*

1 Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan
2 Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca2+, although the difference was not observed without Ca2+. A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Homologous recombinational repair (HRR) is an accurate repair pathway for double-strand breaks (DSBs). Spontaneous chromosomal DSBs accumulate in cells, in which the HRR pathway is defective, indicating that this pathway is essential for chromosome maintenance (Sonoda et al. 2001). The Rad51 protein was identified as a eukaryotic homologue of the bacterial RecA protein, which is an essential enzyme for homologous recombination and HRR (Aboussekhra et al. 1992; Basile et al. 1992; Shinohara et al. 1992, 1993; Morita et al. 1993; Yoshimura et al. 1993). In mice, the Rad51-gene knockout results in early embryonic lethality, probably due to defective HRR (Lim & Hasty 1996; Tsuzuki et al. 1996). In chicken DT40 cells, disrupting the Rad51-gene causes cell death, with the accumulation of spontaneous chromosome breaks (Sonoda et al. 1998). Thus, the Rad51 protein is essential for the HRR pathway in higher eukaryotes.

In bacteria, the RecA protein catalyzes the homologous-pairing and strand-exchange reactions within the HRR pathway (McEntee et al. 1979; Shibata et al. 1979; Cox & Lehman 1981a,b). As expected, in eukaryotes, the Rad51 protein also catalyzes the homologous-pairing and strand-exchange reactions (Sung 1994; Baumann et al. 1996; Maeshima et al. 1996; Gupta et al. 1997). To do so, the Rad51 protein binds to ssDNA produced at DSB sites and forms a helical nucleoprotein filament. The Rad51-ssDNA filament then binds to double-stranded DNA (dsDNA) and forms the ternary complex containing ssDNA, dsDNA and the Rad51 protein. In the ternary complex, a short heteroduplex is formed by the Rad51-mediated homologous pairing. The heteroduplex region is then extended by the subsequent strand-exchange reaction promoted by the Rad51 protein (Sung 1994; Baumann et al. 1996).

Mutations in the Rad51 gene have been identified in several tumors (Kato et al. 2000; Levy-Lahad et al. 2001; Wang et al. 2001; Blasiak et al. 2003; Jakubowska et al. 2003), suggesting that the Rad51-dependent HRR is involved in tumor suppression mechanisms. In addition, the Tyr315 residue of the human Rad51 protein (HsRad51) was found to be constitutively phosphorylated by the BCR/ABL fusion protein, which is derived from the translocation of the c-ABL gene from chromosome 9 to the BCR gene locus on chromosome 22 (Philadelphia chromosome) in leukemia patients (Slupianek et al. 2001). The Tyr315 residue is also phosphorylated by c-Abl in an ATM-dependent manner under ionizing radiation conditions (Chen et al. 1999). Our previous analyses suggested that the Tyr315 residue is located in the monomer–monomer interface of the HsRad51 filament (Conilleau et al. 2004; Takizawa et al. 2004). Therefore, the amino acid residues near position 315 of the HsRad51 protein may also be involved in the monomer–monomer interaction within the HsRad51 filament.

Interestingly, two HsRad51 sequences, HsRad51-K313 and HsRad51-Q313, were deposited in the database (DDBJ accession nos. D14134 and D13804, respectively). The HsRad51-K313 and HsRad51-Q313 proteins have an amino acid difference at position 313, which is located near position 315. The HsRad51-Lys313 residue is highly conserved among the eukaryotic Rad51 proteins. Therefore, the HsRad51-K313 protein may be the conventional HsRad51 protein. On the other hand, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by the Gln residue, is presumed to be an isoform corresponding to a naturally occurring polymorphic variant (Shinohara et al. 1993; Sigurdsson et al. 2001).

In the present study, we purified the HsRad51-K313 and HsRad51-Q313 proteins. We also purified three more HsRad51 mutants, in which the Lys313 residue was replaced by a basic amino acid residue, Arg, an acidic amino acid residue, Glu and a hydrophobic amino acid residue, Met. The biochemical activities of these purified HsRad51 isoforms and mutants were then tested in vitro.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
The strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 isoforms

We purified the HsRad51-K313 and HsRad51-Q313 proteins by a four-step purification method, including nickel-nitrilotriacetic acid (Ni-NTA) agarose column chromatography, removal of the hexahistidine tag from the HsRad51 portion with thrombin protease, spermidine precipitation and MonoQ column chromatography (Fig. 1A, lanes 2 and 3). We then tested the strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 proteins. The {phi}X174 phage circular ssDNA (5386 bases) and the linearized {phi}X174 dsDNA (5386 base pairs) were used as DNA substrates (Fig. 1B) and the reactions were conducted in the presence of human RPA (2 µM) and Ca2+. The Ca2+ ion reportedly stimulated the strand-exchange activity of the HsRad51 protein by preserving the active HsRad51 filament on DNA, through modulation of its ATPase activity (Bugreev & Mazin 2004).


Figure 1
View larger version (39K):
[in this window]
[in a new window]

  		Figure 1  The strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 proteins under the conditions with Ca2+. (A) The purified HsRad51-K313 (lane 2), HsRad51-Q313 (lane 3), HsRad51-R313 (lane 4), HsRad51-E313 (lane 5) and HsRad51-M313 (lane 6) proteins were analyzed by 15% SDS-PAGE with Coomassie Brilliant Blue staining. Lane 1 indicates the molecular mass markers. (B) A schematic diagram of the strand-exchange assay. (C) Time course experiments. The HsRad51 protein was incubated with {phi}X174 circular ssDNA (20 µM) at 37 ºC for 10 min in the Ca2+-containing buffer without KCl. After the addition of RPA, {phi}X174 linear dsDNA (20 µM) was added to initiate the reaction. The reactions were continued for the indicated times. The DNA products were then deproteinized and were separated by 1% agarose gel electrophoresis in 1x TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment (60 min) without the HsRad51 protein. Lanes 2–6 indicate experiments with the HsRad51-K313 protein and lanes 7–11 indicate experiments with the HsRad51-Q313 protein. Reaction times were 0 min (lanes 2 and 7), 15 min (lanes 3 and 8), 30 min (lanes 4 and 9), 60 min (lanes 5 and 10) and 90 min (lanes 6 and 11). (D) A densitometric scanning profile for lane 11 in panel C. Peaks corresponding to the jm intermediates, the nc products, dsDNA and ssDNA are indicated. (E) The band intensities of the jm and nc products in panel C were quantified as the peak volumes of densitometric scans, as shown in panel D. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the reaction time. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (F) The band intensities of the nc products in panel C were quantified and were plotted against the reaction time. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (G) Protein titration experiments. Reactions were conducted for 60 min, according to the same procedure for panel C. Lanes 2–4 indicate experiments with the HsRad51-K313 protein and lanes 6–8 indicate experiments with the HsRad51-Q313 protein. The HsRad51 concentrations were 0 µM (lanes 1 and 5), 2 µM (lanes 2 and 6), 4 µM (lanes 3 and 7) and 6 µM (lanes 4 and 8). (H) The band intensities of the jm and nc products in panel G were quantified as the peak volumes of densitometric scans, as shown in panel D. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the protein concentration. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (I) The band intensities of the nc products in panel G were quantified and were plotted against the protein concentration. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively.

 
In this assay, the HsRad51 protein must first bind ssDNA. Thus, dsDNA was added to the reaction mixture after the HsRad51–ssDNA complex was formed. This reaction order generates the intermediate and complete strand-exchange products, a joint molecule (jm) and a nicked circular DNA (nc) with a displaced ssDNA, respectively (Fig. 1B). To detect these jm and nc products separately, we employed gel conditions in which the jm and nc products were completely separated on a 1% agarose gel without dye and were visualized by SYBR Gold (Invitrogen, Carlsbad, CA) staining after electrophoresis.

Time course and protein titration experiments revealed that the HsRad51-K313 protein catalyzed strand exchange; however, the amounts of the jm intermediates and the nc products formed by the HsRad51-K313 protein were significantly lower than those formed by the HsRad51-Q313 protein under the same reaction conditions with Ca2+ (Fig. 1C–I). Interestingly, in the reactions with the HsRad51-Q313 protein, the jm intermediates, which migrated more slowly than the jm intermediates formed by the HsRad51-K313 protein, were also detected (Fig. 1C,G). This suggests that the ability to form the heteroduplex by strand exchange is different between the HsRad51-K313 and KsRad51-Q313 proteins. These differences in the jm formation between the HsRad51-K313 and HsRad51-Q313 proteins were not obvious when the reactions were performed under conditions with Ca2+ and 200 mM KCl, which enhanced the strand-exchange activity of the HsRad51 protein (Fig. 2A, B, D, E). However, the HsRad51-Q313 protein still exhibited the enhanced nc product formation, as compared to the HsRad51-K313 protein (Fig. 2A,C,D,F). Thus, the HsRad51-Q313 protein possesses the enhanced strand-exchange activity.


Figure 2
View larger version (42K):
[in this window]
[in a new window]

  		Figure 2  The strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 proteins under the conditions with Ca2+ and KCl. (A) Time course experiments. The HsRad51 protein was incubated with {phi}X174 circular ssDNA (20 µM) at 37 °C for 10 min in the Ca2+-containing buffer with 200 mM KCl. After the addition of RPA, {phi}X174 linear dsDNA (20 µM) was added to initiate the reaction. The reactions were continued for the indicated times. The DNA products were then deproteinized and were separated by 1% agarose gel electrophoresis in 1x TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Lane 1 indicates a negative control experiment (60 min) without the HsRad51 protein. Lanes 2–6 indicate experiments with the HsRad51-K313 protein and lanes 7–11 indicate experiments with the HsRad51-Q313 protein. Reaction times were 0 min (lanes 2 and 7), 15 min (lanes 3 and 8), 30 min (lanes 4 and 9), 60 min (lanes 5 and 10) and 90 min (lanes 6 and 11). (B) The band intensities of the jm and nc products in panel A were quantified as the peak volumes of densitometric scans, as shown in panel D of Fig. 1. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the reaction time. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (C) The band intensities of the nc products in panel A were quantified and were plotted against the reaction time. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (D) Protein titration experiments. Reactions were conducted for 60 min, according to the same procedure for panel A. Lanes 2–4 indicate experiments with the HsRad51-K313 protein and lanes 6–8 indicate experiments with the HsRad51-Q313 protein. The HsRad51 concentrations were 0 µM (lanes 1 and 5), 2 µM (lanes 2 and 6), 4 µM (lanes 3 and 7) and 6 µM (lanes 4 and 8). (E) The band intensities of the jm and nc products in panel D were quantified as the peak volumes of densitometric scans, as shown in panel D of Fig. 1. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the protein concentration. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (F) The band intensities of the nc products in panel D were quantified and were plotted against the protein concentration. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively.

 
Consistent with the previous report (Sigurdsson et al. 2001), when the reactions were performed under the conditions without Ca2+ (with ammonium sulfate and spermidine), these differences in the strand-exchange activity between the HsRad51-K313 and HsRad51-Q313 proteins were not obvious (Fig. 3). The Ca2+ ion inhibits the conversion of the active HsRad51-ATP filament into an inactive, ADP-bound form (Bugreev & Mazin 2004). Thus, the HsRad51-Q313 protein may have better filament-formation ability than the HsRad51-K313 protein.


Figure 3
View larger version (60K):
[in this window]
[in a new window]

  		Figure 3  The strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 proteins in the absence of Ca2+. (A) Time course experiments. The HsRad51 protein was incubated with {phi}X174 circular ssDNA (20 µM) at 37 °C for 10 min in the KCl-containing buffer without Ca2+. After the addition of RPA, 100 mM (NH4)2SO4 was added and then {phi}X174 linear dsDNA (20 µM) and spermidine were added to initiate the reaction. The reactions were continued for the indicated times. The DNA products were then deproteinized and were separated by 1% agarose gel electrophoresis in 1x TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Lane 1 indicates a negative control experiment (60 min) without the HsRad51 protein. Lanes 2–6 indicate experiments with the HsRad51-K313 protein and lanes 7–11 indicate experiments with the HsRad51-Q313 protein. Reaction times were 0 min (lanes 2 and 7), 15 min (lanes 3 and 8), 30 min (lanes 4 and 9), 60 min (lanes 5 and 10) and 90 min (lanes 6 and 11). (B) The band intensities of the jm and nc products in panel A were quantified as the peak volumes of densitometric scans, as shown in panel D of Fig. 1. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the reaction time. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. (C) Protein titration experiments. Reactions were conducted for 60 min, according to the same procedure for panel A. Lanes 2-4 indicate experiments with the HsRad51-K313 protein and lanes 6-8 indicate experiments with the HsRad51-Q313 protein. The HsRad51 concentrations were 0 µM (lanes 1 and 5), 2 µM (lanes 2 and 6), 4 µM (lanes 3 and 7) and 6 µM (lanes 4 and 8). (D) The band intensities of the jm and nc products in panel C were quantified as the peak volumes of densitometric scans, as shown in panel D of Fig. 1. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the protein concentration. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively.

 
The ssDNA-binding and ssDNA-dependent ATPase activities of the HsRad51-K313 and HsRad51-Q313 isoforms

Next, we compared the ssDNA-binding activities of the HsRad51-Q313 and HsRad51-K313 proteins. As shown in Fig. 4A, the HsRad51-Q313 protein bound to ssDNA as efficiently as the HsRad51-K313 protein in the absence of Ca2+. Interestingly, in the presence of Ca2+ and a high concentration of the protein (4 µM), both the HsRad51-K313 and HsRad51-Q313 proteins formed large aggregates, which stacked in the wells of the agarose gel (Fig. 4B, lanes 4 and 8). This indicated that the Ca2+ ion actually enhances the polymer formation abilities of the HsRad51-K313 and HsRad51-Q313 proteins with ssDNA. Consistent with its ssDNA-binding ability, the HsRad51-Q313 protein hydrolyzed ATP in a ssDNA-dependent manner, like the HsRad51-K313 protein (Fig. 4C).


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		Figure 4  The ssDNA-binding and ssDNA-dependent ATPase activities. (A and B) The ssDNA-binding experiments. The {phi}X174 circular ssDNA (40 µM) was incubated with the HsRad51-K313 or HsRad51-Q313 protein at 37 °C for 10 min. The samples were analyzed by 0.8% agarose gel electrophoresis in 1x TAE buffer. The bands were visualized by ethidium bromide staining. Protein concentrations were 1 µM (lanes 2 and 6), 2 µM (lanes 3 and 7) and 4 µM (lanes 4 and 8). Lanes 1 and 5 are control experiments without the HsRad51 protein. (A) The ssDNA-binding experiments without Ca2+. (B) The ssDNA-binding experiments with Ca2+. (C) The ATPase activities of the HsRad51-K313 and HsRad51-Q313 proteins. Time course experiments are shown. Closed circles with a solid line and open circles with a broken line indicate experiments with the HsRad51-K313 and HsRad51-Q313 proteins in the presence of ssDNA (40 µM), respectively and closed squares with a solid line and open squares with a broken line indicate experiments with the HsRad51-K313 and HsRad51-Q313 proteins in the absence of ssDNA. (D) Gel filtration analysis. Two independent experiments were performed with 60 and 170 µg of the protein. The elution volumes of the peaks corresponding to the HsRad51-K313 and HsRad51-Q313 proteins are indicated. The elution volume of the human Dmc1 octamer (about 300 kDa) under the same conditions is indicated as a reference.

 
In the gel shift assay, the HsRad51-Q313–ssDNA complex migrated slightly slower than the HsRad51-K313–ssDNA complex (Fig. 4A,B), especially in the presence of Ca2+ (Fig. 4B, lanes 3, 4, 7 and 8). The ssDNA-dependent ATPase activity of the HsRad51-Q313 protein was also slightly higher than that of the HsRad51-K313 protein in the presence of 40 µM ssDNA (Fig. 4C). When the ssDNA concentration was reduced to 10 µM, these differences between the HsRad51-Q313 and HsRad51-K313 proteins in the ssDNA-dependent ATPase activity were still observed (data not shown). In the absence of ssDNA, the HsRad51-Q313 protein eluted slightly faster than the HsRad51-K313 protein from the Superdex 200 gel filtration column, at two different concentrations (60 and 170 µg) (Fig. 4D). Although the differences between the HsRad51-Q313 and HsRad51-K313 proteins in the ssDNA-binding, ssDNA-dependent ATPase and polymer formation activities are not significant, these small variations implied that the HsRad51-Q313 protein may have better filament-formation ability than the HsRad51-K313 protein.

The dsDNA-unwinding abilities of the HsRad51-K313 and HsRad51-Q313 isoforms

To test whether the HsRad51-Q313 protein has better filament-formation ability than the HsRad51-K313 protein, we performed the dsDNA unwinding assay, which is a more sensitive assay to detect the differences in the filament-formation activity of Rad51 on dsDNA (Fig. 5C). Electron microscopic visualization revealed that both the HsRad51-Q313 and HsRad51-K313 proteins formed nucleoprotein filaments on dsDNA (Fig. 5A,B). The Rad51 protein is known to extensively unwind dsDNA in the Rad51-dsDNA filament, in an ATP-dependent manner (Benson et al. 1994). When covalently closed dsDNA was used as substrate, the ATP-dependent dsDNA unwinding by the Rad51 protein resulted in highly negatively supercoiled DNA (form X). The production of form X depends on the Rad51-filament formation, in which the dsDNA is unwound by the cooperative binding of the Rad51 protein (Fig. 5C).


Figure 5
View larger version (82K):
[in this window]
[in a new window]

  		Figure 5  The dsDNA-unwinding activity. (A and B) Electron microscopic images of the HsRad51-K313-dsDNA (A) and HsRad51-Q313-dsDNA (B) complexes. The HsRad51 filaments formed on a nicked circular pUC119 dsDNA. Six images are presented for each HsRad51 protein. The black bar denotes 100 nm. (C) Schematic representation of the topological assay for dsDNA unwinding. Supercoiled dsDNA is relaxed by eukaryotic topoisomerase I. The dsDNA is unwound by the HsRad51 binding and compensatory positive supercoils (+) are introduced. Highly negative supercoiled DNA (form X) is formed by relaxation of the dsDNA bound to the HsRad51 protein by topoisomerase I, followed by deproteination. (D) The dsDNA-unwinding activities of the HsRad51-K313 and HsRad51-Q313 proteins. The {phi}X174 dsDNA (form I) containing nicked circular DNA (form II) was incubated with the HsRad51 protein at 23 °C for 30 min. Topoisomerase I (10 units) was then added and the reaction mixture was incubated at 23 °C for 60 min. The reaction was stopped by adding 1 µL of 1% SDS, followed by the immediate addition of 1 µL of 19.7 mg/mL proteinase K. The reaction products were separated on a 1% agarose gel in 1x TAE buffer and the bands were visualized by ethidium bromide staining. Lane 1 indicates the supercoiled DNA (form I DNA) and lane 2 indicates the control experiment without the HsRad51 protein. The experiments with the HsRad51-K313 protein (lanes 3, 5 and 7) and the HsRad51-Q313 protein (lanes 4, 6 and 8) are presented. The HsRad51 concentrations were 4 µM (lanes 3 and 4), 6 µM (lanes 5 and 6) and 8 µM (lanes 7 and 8). (E) The form X products, generated using the same reaction conditions as in panel D, were separated on a 1% agarose gel with 10 µg/mL chloroquine in 1x TAE buffer at 3.3 V/cm for 20 h. The bands were visualized by ethidium bromide staining.

 
As shown in Fig. 5D,E, the form X DNA produced by the HsRad51-Q313 protein clearly migrated faster than that produced by the HsRad51-K313 protein, indicating that the dsDNA-unwinding activity of the HsRad51-Q313 protein is higher than that of the HsRad51-K313 protein. The length of the unwound dsDNA region may directly correlate with the dsDNA region where the Rad51 protein forms filaments. Therefore, these results suggest that the HsRad51-Q313 protein possesses better filament-formation ability than the HsRad51-K313 protein. Another possibility is that the ability of the HsRad51-Q313 protein to unwind the double helix may be augmented, without enhanced filament-formation ability. However, we prefer a model in which the increased filament-formation ability of the HsRad51-Q313 protein is responsible for the dsDNA-unwinding enhancement, because the HsRad51-Q313 protein formed larger polymers than the HsRad51-K313 protein in the absence of DNA (Fig. 4D).

Mutational analysis of the HsRad51-313 residue

We designed three HsRad51 mutants, R313, E313 and M313, in which the Lys313 residue was replaced by Arg, Glu and Met, respectively. Arg, a positively charged hydrophilic residue, may not change the hydrophobic and electrostatic environments around the Lys313 residue. Glu, a negatively charged hydrophilic residue, may change the electrostatic environment. Met, a hydrophobic residue, may change the hydrophobic and electrostatic environments. All three HsRad51 mutants were purified by a four-step purification method, as described above (Fig. 1A, lanes 4–6).

We then tested the strand-exchange activities of these HsRad51 mutants in the presence of Ca2+ and found that the HsRad51-E313 mutant exhibited enhanced strand-exchange activity (Fig. 6A, lanes 5–7 and B,C), like the HsRad51-Q313 isoform (Fig. 6A, lanes 11–13 and B,C). In contrast, the strand-exchange activity of the HsRad51-R313 mutant is the same as that of the HsRad51-K313 protein (Fig. 6A, lanes 14–16 and B,C). The HsRad51-M313 mutant exhibited moderately enhanced strand-exchange activity, as compared to the HsRad51-K313 protein (Fig. 6A, lanes 8–10 and B,C). As shown in Fig. 7A,B, the dsDNA-unwinding activities of the HsRad51-Q313, HsRad51-E313 and HsRad51-M313 proteins (lanes 4, 6 and 7) were better than those of the HsRad51-K313 and HsRad51-R313 proteins (lanes 3 and 5). Therefore, these results indicate that a positive charge (Lys and Arg), but not a negative (Glu) or neutral (Gln and Met) charge, at position 313 of the HsRad51 protein reduces the strand-exchange ability, probably due to its diminished filament-formation ability.


Figure 6
View larger version (39K):
[in this window]
[in a new window]

  		Figure 6  The strand-exchange activities of the HsRad51-E313, HsRad51-M313 and HsRad51-R313 mutants. (A) The reactions were performed in the presence of Ca2+ (without KCl). Lane 1 indicates the control experiment without the HsRad51 protein. Lanes 2–4 indicate control experiments with the HsRad51-K313 protein. Lanes 5–7, lanes 8–10, lanes 11–13 and lanes 14–16 indicate experiments with the HsRad51-E313 protein, the HsRad51-M313 protein, the HsRad51-Q313 protein and the HsRad51-R313 protein, respectively. The HsRad51 concentrations were 0 µM (lane 1), 2 µM (lanes 2, 5, 8, 11 and 14), 4 µM (lanes 3, 6, 9, 12 and 15) and 6 µM (lanes 4, 7, 10, 13 and 16). (B) The band intensities of the jm and nc products in panel A were quantified as the peak volumes of densitometric scans, as shown in panel D of Fig. 1. The band intensities of the gel containing lanes 11–16 were normalized to those of the gel containing lanes 1–10. The ratios (%) of the products relative to the sum total of the peak volumes of all DNAs were plotted against the protein concentration. (C) The band intensities of the nc products in panel A were quantified and were plotted against the protein concentration. The band intensities of the gel containing lanes 11–16 were normalized to those of the gel containing lanes 1–10. Closed circles with a solid line and open circles with a broken line indicate the HsRad51-K313 and HsRad51-Q313 proteins, respectively. Closed squares with a solid line, open squares with a broken line and open triangles with a broken line indicate the HsRad51-E313, HsRad51-M313 and HsRad51-R313 proteins, respectively.

 

Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		Figure 7  The dsDNA-unwinding activities of the HsRad51-E313, HsRad51-M313 and HsRad51-R313 mutants. The {phi}X174 dsDNA (form I) containing nicked circular DNA (form II) was incubated with the HsRad51 protein at 23 °C for 30 min. Topoisomerase I (10 units) was then added and the reaction mixture was incubated at 23 °C for 60 min. The reaction was stopped by adding 1 µL of 1% SDS, followed by the immediate addition of 1 µL of 19.7 mg/mL proteinase K. (A) The reaction products were separated on a 1% agarose gel in 1x TAE buffer at 3.3 V/cm for 20 h and the bands were visualized by ethidium bromide staining. Lane 1 indicates the supercoiled DNA (form I DNA) and lane 2 indicates the control experiment without the HsRad51 protein. The experiments with the HsRad51-K313 protein (lane 3), the HsRad51-Q313 protein (lane 4), the HsRad51-R313 protein (lane 5), the HsRad51-E313 protein (lane 6) and the HsRad51-M313 protein (lane 7) are presented. The HsRad51 concentration was 8 µM. (B) The form X products, generated under the same reaction conditions as in panel A, were separated on a 1% agarose gel with 10 µg/mL chloroquine in 1x TAE buffer at 3.3 V/cm for 20 h. The bands were visualized by ethidium bromide staining.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Two HsRad51 sequences, HsRad51-K313 and HsRad51-Q313 (DDBJ accession nos.: D14134 and D13804, respectively), were deposited in the database. The HsRad51-K313 protein has the Lys313 residue, which is highly conserved among the eukaryotic Rad51 proteins. Thus, the HsRad51-K313 type may be the conventional Rad51 protein. On the other hand, the HsRad51-Q313 protein has a Gln residue at position 313. The HsRad51-Q313 type is presumed to be an isoform corresponding to a naturally occurring polymorphic variant (Shinohara et al. 1993; Sigurdsson et al. 2001).

In the present study, we first compared the strand-exchange activities of the HsRad51-K313 and HsRad51-Q313 proteins under the conditions with Ca2+ and found that the HsRad51-Q313 protein exhibited enhanced strand-exchange activity, as compared to that of the conventional HsRad51-K313 protein, probably through its enhanced filament-formation ability. In agreement with a previous report (Sigurdsson et al. 2001), the difference in the strand-exchange activity between the HsRad51-K313 and HsRad51-Q313 proteins was not observed, when the reactions were conducted under conditions without Ca2+ (with ammonium sulfate and spermidine). Thus, the Ca2+ ion is an essential factor to reveal the difference in the strand-exchange activity between the HsRad51-K313 and HsRad51-Q313 proteins. The Ca2+ ion reportedly keeps the active HsRad51-ATP-ssDNA filament from dissociating into an inactive ADP-bound form, by modulating its ATPase activity (Bugreev & Mazin 2004). Hence, in the presence of Ca2+, it may become possible to evaluate the strand-exchange activity of the active HsRad51-filament species, because the conversion from the active HsRad51 filament into the inactive form is inhibited.

The enhanced homologous-recombination activity, which may be promoted by the HsRad51-Q313 protein, may cause chromosome aberrations, such as translocations and inversions between homologous sequences. Actually, aneuploidy and multiple chromosomal rearrangements were promoted, when the HsRad51 protein was over-expressed in mammalian cells (Richardson et al. 2004). Thus, the proper control of the homologous-recombination activity is important for the maintenance of genome integrity. Furthermore, enhanced HsRad51 expression was observed in human soft tissue sarcoma cells (Hannay et al. 2007) and the BCR/ABL found in leukemia cells augments the HsRad51-dependent recombination frequency in cells (Slupianek et al. 2001). Such enhancement of the HsRad51 activity has the potential to accelerate malignant transformation and to contribute to chemoresistance in tumor cells. The rare HsRad51-Q313 isoform, which has the enhanced strand-exchange activity, may increase the frequency of chromosome aberrations and chemoresistance in tumor cells by inappropriate recombination in human cells.

The Lys313 residue of the HsRad51 protein is located near the Tyr315 residue, which is constitutively phosphorylated by the BCR/ABL tyrosine kinase in leukemia cells (Slupianek et al. 2001). Our previous analyses suggested that the HsRad51-Tyr315 residue is directly involved in the monomer–monomer interface of the HsRad51 filament (Conilleau et al. 2004), implying that the HsRad51-Lys313 residue may also function in the monomer–monomer interactions of filament formation. Thus, we performed a mutational analysis of the HsRad51-Lys313 residue. The HsRad51-R313, HsRad51-E313 and HsRad51-M313 mutants, in which the Lys313 residue was replaced by Arg, Glu and Met, respectively, were prepared. These Arg, Glu and Met residues were selected as representatives of basic, acidic and hydrophobic amino acid residues, respectively. We found that a positive charge (Lys and Arg), but not a negative charge (Glu), at position 313 of the HsRad51 protein reduced the strand-exchange ability, probably due to its reduced filament-formation ability. Thus, the electrostatic environment around the monomer–monomer interface of the HsRad51 filament plays an important role in the regulation of the HsRad51 function.

To understand the function of the HsRad51-Lys313 residue in the monomer–monomer interaction within the HsRad51 filament, we superimposed the HsRad51-ATPase domain structure (Pellegrini et al. 2002) on the MvRadA filament structure (Wu et al. 2004), which has a helical pitch close to that of the putative active Rad51 filament. In this model, however, the HsRad51-Lys313 residue did not directly contact the neighboring HsRad51 protomer (data not shown). Since the HsRad51-Lys313 residue is predicted to be located near the monomer–monomer interface of the HsRad51 filament, a substitution at position 313 may allosterically affect the monomer–monomer interaction within the HsRad51 filament. The other possibility is that the HsRad51-Lys313 residue faces the monomer–monomer interface in an unpredictable manner in the active filament on ssDNA. Further biochemical and structural analyses of the active HsRad51 filament are awaited.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Preparation of the HsRad51-K313, HsRad51-Q313, HsRad51-R313, HsRad51-E313 and HsRad51-M313 proteins

The pET15b expression vectors, including the HsRad51 genes inserted at the NdeI site, were constructed using a Quik-Change kit (Stratagene, La Jolla, CA). The hexahistidine-tagged HsRad51 proteins were over-expressed in the E. coli strain JM109 (DE3), which also carried an expression vector for the minor tRNAs (Codon(+)RIL, Novagen). The HsRad51 proteins expressed in the E. coli strain were purified by the four-step method, as described previously (Matsuo et al. 2006). In this method, the purified HsRad51 protein did not include a hexahistidine tag. Protein concentrations were determined using the Bradford method (Bradford 1976), with bovine serum albumin (Pierce, Woburn, MA) as the standard protein.

DNAs

The {phi}X174 phage ssDNA and dsDNA used in the DNA-binding, strand-exchange, dsDNA-unwinding assays were purchased from New England Biolabs. All DNA concentrations are expressed in moles of nucleotides.

Assay for strand exchange

The {phi}X174 circular ssDNA (20 µM) was incubated with the HsRad51 protein at 37 °C for 10 min, in 10 µL of 28 mM HEPES buffer (pH 7.5), containing 60 mM NaCl, 0.04 mM EDTA, 0.8 mM 2-mercaptoethanol, 4% glycerol, 1 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.1 mg/mL bovine serum albumin, 2 mM CaCl2, 20 mM creatine phosphate and 75 µg/mL creatine kinase. When the reactions were conducted in the presence of KCl, 200 mM KCl was added in the reaction mixture. After this incubation, 2 µM RPA was added to the reaction mixture, which was incubated at 37 °C for 10 min. The reactions were then initiated by the addition of 20 µM {phi}X174 linear dsDNA and were continued for the indicated times. For the experiments without CaCl2, 100 mM KCl was added to the reaction mixture and 100 mM (NH4)2SO4 was added after the addition of 2 µM RPA. The reactions were then initiated by the addition of 20 µM {phi}X174 linear dsDNA and 4 mM spermidine and were continued for the indicated times. The reactions were stopped by the addition of 0.1% SDS and 1.97 mg/mL proteinase K (Roche Applied Science) and were further incubated at 37 °C for 20 min. After adding sixfold loading dye, the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1x TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining.

dsDNA unwinding assay

The HsRad51 protein (4, 6, and 8 µM) was incubated with the supercoiled dsDNA ({phi}X174; 20 µM) in 10 µL standard reaction buffer, containing 28 mM HEPES (pH 7.5), 60 mM NaCl, 0.04 mM EDTA, 0.8 mM 2-mercaptoethanol, 4% glycerol, 1 mM MgCl2, 1 mM DTT, 1 mM ATP and 0.1 mg/mL BSA, at 23 °C for 30 min. After incubation, 10 units of wheat germ topoisomerase I (Promega, Madison, WI) were added and the dsDNA was allowed to relax for 1 h at 23 °C. The reaction was stopped by adding 1 µL of 1% SDS, followed by the immediate addition of 1 µL in 19.7 mg/mL proteinase K (Roche Applied Science, Basel Switzerland). The reaction mixtures were further incubated at 23 °C for 20 min. The reaction mixtures were then separated on a 1% agarose gel with or without 10 µg/mL chloroquine in 1x TAE buffer (40 mM Tris–acetate and 1 mM EDTA) at 3.3 V/cm for 20 h. The bands were visualized by ethidium bromide staining.

ATPase activity

The HsRad51-K313 (5 µM) or HsRad51-Q313 (5 µM) proteins was incubated with 1 mM ATP (Roche Applied Science) in 28 mM HEPES buffer (pH 7.5), containing 60 mM NaCl, 1 mM MgCl2, 4% glycerol, 0.04 mM EDTA, 0.8 mM 2-mercaptoethanol, 1 mM dithiothreitol and 0.1 mg/mL bovine serum albumin, in the presence or absence of ssDNA. In the ssDNA-dependent reaction, the {phi}X174 circular ssDNA (40 µM) was used as the substrate. The reaction was performed at 37 °C. After a 10 min pre-incubation in the absence of ATP, the reaction was initiated by adding 1 mM ATP. At each indicated time, a 20 µL aliquot of the reaction mixture was removed and mixed with 30 µL of 100 mM EDTA, to quench the reaction. The amount of inorganic phosphate released was determined by a colorimetric assay. Briefly, a 500-µL aliquot of a malachite green solution [0.034% (w/v) malachite green oxalate, 1.05% (w/v) hexaammonium heptamolybdate tetrahydrate and 0.1% (w/v) polyvinyl alcohol in 1 M HCl] was mixed with 50 µL of sample solution (i.e. the reaction mixture quenched with EDTA). After 1 min, 50 µL of 34% (w/v) sodium citrate dihydrate was added to stop further color development. The absorbance at 655 nm was measured with a 96-well micro plate reader (Bio-Rad, Hercules, CA). A 1-mg/mL phosphate ion standard solution (Wako Pure Chemicals, Osaka, Japan) was used to prepare the phosphate standards.

Assays for ssDNA binding

The {phi}X174 circular ssDNA (40 µM) was mixed with the HsRad51-K313 or HsRad51-Q313 protein in 10 µL of 24 mM HEPES buffer (pH 7.5), containing 30 mM NaCl, 1 mM MgCl2, 2% glycerol, 0.02 mM EDTA, 0.4 mM 2-mercaptoethanol, 1 mM dithiothreitol, 0.1 mg/mL bovine serum albumin and 1 mM ATP in the presence or absence of 2 mM CaCl2. The reaction mixtures were incubated at 37 °C for 10 min and were then analyzed by 0.8% agarose gel electrophoresis in 1x TAE buffer (40 mM Tris–acetate and 1 mM EDTA) at 3.3 V/cm for 2 h. The bands were visualized by ethidium bromide staining.

Gel filtration

The HsRad51-K313 and HsRad51-Q313 proteins (60 or 170 µg) were analyzed by Superdex 200 HR 10/30 (GE Healthcare Bio-Sciences, Chicago, IL) gel filtration chromatography. The elution buffer contained 20 mM HEPES–NaOH (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 2 mM 2-mercaptoethanol and 10% glycerol and the flow rate was 0.3 mL/min.

Electron microscopy

The HsRad51-K313 or HsRad51-Q313 protein (1 µM) was mixed with pUC119 form II DNA (1 µM) in 20 mM triethanolamine, 1 mM Mg–acetate and 1 mM ATP{gamma}S and was incubated for 10 min at 37 °C. Samples (3 µL) were adsorbed on a carbon grid and stained with 2% uranium acetate. The samples were examined with a JEOL JEM2000FX electron microscope.


   Acknowledgements
 
We thank Drs T. Shibata and S. Ikawa for the electron microscopic analysis and Dr M. S. Wold (University of Iowa) for providing the human RPA expression vector. This work was supported by Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.K. and I.S. were supported by the program, ‘Establishment of Consolidated Research Institute for Advanced Science and Medical Care,’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Fumio Hanaoka

* Correspondence: E-mail: kurumizaka{at}waseda.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Aboussekhra, A., Chanet, R., Adjiri, A. & Fabre, F. (1992) Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 12, 3224–3234.[Abstract/Free Full Text]

Basile, G., Aker, M. & Mortimer, R.K. (1992) Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol. Cell. Biol. 12, 3235–3246.[Abstract/Free Full Text]

Baumann, P., Benson, F.E. & West, S.C. (1996) Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766.[CrossRef][Medline]

Benson, F.E., Stasiak, A. & West, S.C. (1994) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771.[Medline]

Blasiak, J., Przybylowska, K., Czechowska, A., Zadrozny, M., Pertynski, T., Rykala, J., Kolacinska, A., Morawiec, Z. & Drzewoski, J. (2003) Analysis of the G/C polymorphism in the 5'-untranslated region of the RAD51 gene in breast cancer. Acta Biochim. Pol. 50, 249–253.[Medline]

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.[CrossRef][Medline]

Bugreev, D.V. & Mazin, A. (2004) Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl. Acad. Sci. USA 101, 9988–9993.[Abstract/Free Full Text]

Chen, G., Yuan, S.S., Liu, W., Xu, Y., Trujillo, K., Song, B., Cong, F., Goff, S.P., Wu, Y., Arlinghaus, R., Baltimore, D., Gasser, P.J., Park, M.S., Sung, P. & Lee, E.Y. (1999) Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl. J. Biol. Chem. 274, 12748–12752.[Abstract/Free Full Text]

Conilleau, S., Takizawa, Y., Tachiwana, H., Fleury, F., Kurumizaka, H. & Takahashi, M. (2004) Location of tyrosine 315, a target for phosphorylation by cAbl tyrosine kinase, at the edge of the subunit–subunit interface of the human Rad51 filament. J. Mol. Biol. 339, 797–804.[CrossRef][Medline]

Cox, M.M. & Lehman, I.R. (1981a) recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc. Natl. Acad. Sci. USA 78, 3433–3437.[Abstract/Free Full Text]

Cox, M.M. & Lehman, I.R. (1981b) Directionality and polarity in recA protein-promoted branch migration. Proc. Natl. Acad. Sci. USA 78, 6018–6022.[Abstract/Free Full Text]

Gupta, R.C., Bazemore, L.R., Golub, E.I. & Radding, C.M. (1997) Activities of human recombination protein Rad51. Proc. Natl. Acad. Sci. USA 94, 463–468.[Abstract/Free Full Text]

Hannay, J.A., Liu, J., Zhu, Q.S., Bolshakov, S.V., Li, L., Pisters, P.W., Lazar, A.J., Yu, D., Pollock, R.E. & Lev, D. (2007) Rad51 overexpression contributes to chemoresistance in human soft tissue sarcoma cells: a role for p53/activator protein 2 transcriptional regulation. Mol. Cancer Ther. 6, 1650–1660.[Abstract/Free Full Text]

Jakubowska, A., Narod, S.A., Goldgar, D.E., Mierzejewski, M., Masojc, B., Nej, K., Huzarska, J., Byrski, T., Górski, B. & Lubinski, J. (2003) Breast cancer risk reduction associated with the RAD51 polymorphism among carriers of the BRCA1 5382insC mutation in Poland. Cancer Epidemiol. Biomarkers Prev. 12, 457–459.[Abstract/Free Full Text]

Kato, M., Yano, K., Matsuo, F., Saito, H., Katagiri, T., Kurumizaka, H., Yoshimoto, M., Kasumi, F., Akiyama, F., Sakamoto, G., Nagawa, H., Nakamura, Y. & Miki, Y. (2000) Identification of Rad51 alteration in patients with bilateral breast cancer. J. Hum. Genet. 45, 133–137.[CrossRef][Medline]

Levy-Lahad, E., Lahad, A., Eisenberg, S., Dagan, E., Paperna, T., Kasinetz, L., Catane, R., Kaufman, B., Beller, U., Renbaum, P. & Gershoni-Baruch, R. (2001) A single nucleotide polymorphism in the RAD51 gene modifies cancer risk in BRCA2 but not BRCA1 carriers. Proc. Natl. Acad. Sci. USA 98, 3232–3236.[Abstract/Free Full Text]

Lim, D.-S. & Hasty, P. (1996) A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol. Cell. Biol. 16, 7133–7143.[Abstract]

Maeshima, K., Morimatsu, K. & Horii, T. (1996) Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction. Genes Cells 1, 1057–1068.[Abstract]

Matsuo, Y., Sakane, I., Takizawa, Y., Takahashi, M. & Kurumizaka, H. (2006) Roles of the human Rad51 L1 and L2 loops in DNA binding. FEBS J. 273, 3148–3159.[CrossRef][Medline]

McEntee, K., Weinstock, G.M. & Lehman, I.R. (1979) Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc. Natl. Acad. Sci. USA 76, 2615–2619.[Abstract/Free Full Text]

Morita, T., Yoshimura, Y., Yamamoto, A., Murata, K., Mori, M., Yamamoto, H. & Matsushiro, A. (1993) A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 genes. Proc. Natl. Acad. Sci. USA. 90, 6577–6580.[Abstract/Free Full Text]

Pellegrini, L., Yu, D.S., Lo, T., Anand, S., Lee, M., Blundell, T.L. & Venkitaraman, A.R. (2002) Insights into DNA recombination from the structure of a RAD51–BRCA2 complex. Nature 420, 287–293.[CrossRef][Medline]

Richardson, C., Stark, J.M., Ommundsen, M. & Jasin, M. (2004) Rad51 overexpression promotes alternative double-strand break repair pathways and genome instability. Oncogene 23, 546–553.[CrossRef][Medline]

Shibata, T., DasGupta, C., Cunningham, R.P. & Radding, C.M. (1979) Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. Proc. Natl. Acad. Sci. USA 76, 1638–1642.[Abstract/Free Full Text]

Shinohara, A., Ogawa, H. & Ogawa, T. (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell 69, 457–470.[CrossRef][Medline]

Shinohara, A., Ogawa, H., Matsuda, Y., Ushio, N., Ikeo, K. & Ogawa, T. (1993) Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA. Nat. Genet. 4, 239–243.[CrossRef][Medline]

Sigurdsson, S., Trujillo, K., Song, B., Stratton, S. & Sung, P. (2001) Basis for avid homologous DNA strand exchange by human Rad51 and RPA. J. Biol. Chem. 276, 8798–8806.[Abstract/Free Full Text]

Slupianek, A., Schmutte, C., Tombline, G., Nieborowska-Skorska, M., Hoser, G., Nowicki, M.O., Pierce, A.J., Fishel, R. & Skorski, T. (2001) BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance. Mol. Cell 8, 795–806.[CrossRef][Medline]

Sonoda, E., Sasaki, M.S., Buerstedde, J.-M., Bezzubova, O., Shinohara, A., Ogawa, H., Takata, M., Yamaguchi-Iwai, Y. & Takeda, S. (1998) Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death. EMBO J. 17, 598–608.[CrossRef][Medline]

Sonoda, E., Takata, M., Yamashita, Y.M., Morrison, C. & Takeda, S. (2001) Homologous DNA recombination in vertebrate cells. Proc. Natl. Acad. Sci. USA 98, 8388–8394.[Abstract/Free Full Text]

Sung, P. (1994) Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243.[Abstract/Free Full Text]

Takizawa, Y., Kinebuchi, T., Kagawa, W., Yokoyama, S., Shibata, T. & Kurumizaka, H. (2004) Mutational analyses of the human Rad51-Tyr315 residue, a site for phosphorylation in leukaemia cells. Genes Cells 9, 781–790.[Abstract/Free Full Text]

Tsuzuki, T., Fujii, Y., Sakumi, K., Tominaga, Y., Nakao, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y. & Morita, T. (1996) Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA 93, 6236–6240.[Abstract/Free Full Text]

Wang, W.W., Spurdle, A.B., Kolachana, P., et al. (2001) A single nucleotide polymorphism in the 5' untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers. Cancer Epidemiol. Biomarkers Prev. 10, 955–960.[Abstract/Free Full Text]

Wu, Y., He, Y., Moya, I.A., Qian, X. & Luo, Y. (2004) Crystal structure of archaeal recombinase RADA: snapshot of its extended comformation. Mol. Cell 15, 423–435.[CrossRef][Medline]

Yoshimura, Y., Morita, T., Yamamoto, A. & Matsushiro, A. (1993) Cloning and sequence of the human RecA-like gene cDNA. Nucleic Acids Res. 21, 1665.[Free Full Text]

Received: 16 June 2007
Accepted: 23 October 2007




This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
I. Sakane, C. Kamataki, Y. Takizawa, M. Nakashima, S. Toki, H. Ichikawa, S. Ikawa, T. Shibata, and H. Kurumizaka
Filament formation and robust strand exchange activities of the rice DMC1A and DMC1B proteins
Nucleic Acids Res., August 1, 2008; 36(13): 4266 - 4276.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ishida, T.
Right arrow Articles by Kurumizaka, H.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Ishida, T.
Right arrow Articles by Kurumizaka, H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS