In addition to these RAD51-binding proteins, we previously reported that human EVL binds directly to RAD51 and RAD51B, and stimulates RAD51-medi-ated homologous pairing and strand exchan
Trang 1homologous recombination
Motoki Takaku, Shinichi Machida, Shugo Nakayama, Daisuke Takahashi and Hitoshi Kurumizaka
Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan
Introduction
Chromosomal DNA is constantly exposed to various
DNA-damaging agents, including ionizing radiation,
crosslinking reagents, and oxidative stress A
double-strand break (DSB), which is induced by such
DNA-damaging agents and⁄ or failure of DNA replication,
results in chromosome aberrations and tumorigenesis,
if it is not properly repaired [1–3] Homologous
recom-binational repair (HRR) is a major pathway for the
repair of DSBs in higher eukaryotes [4]
RAD51, an essential enzyme for the HRR pathway,
promotes the homologous pairing and strand exchange
reactions, which are key steps in homologous
recombi-nation To efficiently promote the homologous pairing
and strand exchange reactions, RAD51 requires several
auxiliary factors, which directly or indirectly interact
with RAD51 [5,6] In humans, RAD52, RAD54,
RAD54B, RAD51B–RAD51C, RAD51AP1, BRCA2 and PSF have been reported to be RAD51-binding proteins that modulate the homologous pairing and⁄ or strand exchange reactions promoted by RAD51 [7–10]
In addition to these RAD51-binding proteins, we previously reported that human EVL binds directly to RAD51 and RAD51B, and stimulates RAD51-medi-ated homologous pairing and strand exchange in vitro [11] Human EVL is a member of the ENA⁄ VASP family, which is involved in actin-remodeling processes, and is composed of 418 amino acids forming three dis-tinct domains, EVH1, Pro-rich, and EVH2 [12] Two other ENA⁄ VASP family proteins, MENA and VASP, also contain these three domains, but the significance
of these domains in homologous recombination has not been elucidated
Keywords
DSB; ENA/VASP; EVL; homologous
recombination; RAD51
Correspondence
H Kurumizaka, Laboratory of Structural
Biology, Graduate School of Advanced
Science and Engineering, Waseda
University, 2-2 Wakamatsu-cho, Shinjuku-ku,
Tokyo 162-8480, Japan
Fax: +81 3 5367 2820
Tel: +81 3 5369 7315
E-mail: kurumizaka@waseda.jp
(Received 3 June 2009, revised 3 August
2009, accepted 6 August 2009)
doi:10.1111/j.1742-4658.2009.07265.x
EVL is a member of the ENA⁄ VASP family, which is involved in actin-remodeling processes Previously, we reported that human EVL directly interacts with RAD51, which is an essential protein in the homologous recombinational repair of DNA double-strand breaks, and stimulates RAD51-mediated recombination reactions in vitro To identify the EVL domain required for the recombination function, we purified the EVL fragments EVL(1–221) and EVL(222–418), which contain the EVH1 and Pro-rich domains and the EVH2 domain, respectively We found that EVL(222–418) possesses DNA-binding and RAD51-binding activities, and also stimulates RAD51-mediated homologous pairing In contrast, EVL(1–221) did not exhibit any of these activities Therefore, the EVH2 domain, which is highly conserved among the ENA⁄ VASP family proteins, may be responsible for the recombination function of EVL
Structured digital abstract
l MINT-7239394 : EVL (uniprotkb: Q9UI08 ) binds ( MI:0407 ) to RAD51 (uniprotkb: Q06609 ) by pull down ( MI:0096 )
Abbreviations
DSB, double-strand break; HRR, homologous recombinational repair; MMC, mitomycin C.
Trang 2To identify the functional domain for the
recombi-nation-related activity of EVL, we purified two EVL
fragments, corresponding to the EVH1 and Pro-rich
domains and the EVH2 domain, as well as the
full-length EVL protein We found that the EVH2
domain, but not the EVH1 and Pro-rich domains, is
responsible for the recombination-related activity of
EVL
Results
Preparation of EVL(1–221) and EVL(222–418)
To identify the functional domain responsible for the
recombination activity of human EVL, we purified two
EVL fragments, EVL(1–221) and EVL(222–418),
con-taining amino acids 1–221 and 222–418, respectively
(Fig 1A) EVL(1–221) contained both the EVH1 and
Pro-rich domains, and EVL(222–418) contained the
EVH2 domain (Fig 1A) EVL(1–221) and EVL(222–
418) were purified as recombinant proteins by a five-step procedure (Fig 1B,C) In this procedure, the His6 tag was removed by thrombin protease treatment, and the proteins without the His6 tag consequently migrated slightly faster than the His6-tagged proteins upon SDS⁄ PAGE (Fig 1B,C, lane 6) EVL(1–221) and EVL(222–418) were further purified by gel filtration chromatography and MonoS chromatography (Fig 1B,C, lanes 7 and 8)
The EVH2 domain is responsible for the DNA-binding activity of EVL
We first tested the ssDNA-binding and dsDNA-bind-ing activities of EVL(1–221) and EVL(222–418), because the DNA-binding activity of EVL was reported previously [11] As shown in Fig 2A,B, EVL(222–418) bound to both ssDNA and dsDNA, although its activity was somewhat reduced as com-pared with the DNA-binding activity of full-length EVL The apparent EVH2⁄ nucleotide ratio in EVH2– ssDNA binding was about 1 : 20 In contrast, EVL(1–221) bound to neither ssDNA nor dsDNA (Fig 2A,B) A competitive binding assay with ssDNA and dsDNA revealed that EVL(222–418), like full-length EVL [11], bound preferentially to ssDNA rather than dsDNA (Fig 2C) Therefore, the EVH2 domain is responsible for the DNA-binding activity
of EVL
The EVH2 domain binds RAD51 Previously, we found that EVL directly binds to RAD51 [11], which is an essential protein for the HRR pathway in eukaryotes We then tested whether EVL(1–221) and EVL(222–418) could bind to RAD51
To do this, we performed Ni2+–nitrilotriacetic acid bead pull-down assays with the His6-tagged EVL, EVL(1–221) and EVL(222–418) proteins (Fig 3A) As shown in Fig 3A (lane 6) and Fig 3B, His6-tagged EVL copelleted with RAD51, indicating that this assay could be useful for the detection of EVL–RAD51 binding Interestingly, a significant amount of RAD51 was captured by the Ni2+–nitrilotriacetic acid agarose beads in the presence of His6-tagged EVL(222–418) (Fig 3A, lane 8, and Fig 3B) Careful titration experi-ments revealed that the RAD51⁄ EVL(222–418) bind-ing stoichiometry was 1 : 2 (Fig 3C,D) In contrast, only background levels of His6-tagged EVL(1–221) copelleted with RAD51 (Fig 3A, lane 7, and Fig 3B) These results indicate that the EVH2 domain, but not the EVH1 and Pro-rich domains, directly binds to RAD51
B
A
C
Fig 1 Purification of EVL(1–221) and EVL(222–418) (A) Schematic
representation of the EVL fragments Boxes denoted as EVH1,
P-rich and EVH2 represent regions corresponding to the EVH1,
Pro-rich and EVH2 domains, respectively (B) Purification of
EVL(1–221) (C) Purification of EVL(222–418) Proteins from each
purification step were analyzed by 15% SDS ⁄ PAGE with
Coomas-sie Brilliant Blue staining Lane 1: molecular mass markers Lanes 2
and 3: whole cell lysates before and after induction with isopropyl
thio-b- D -galactoside (IPTG), respectively Lanes 4–8: samples from
the peak Ni 2+ –nitrilotriacetic acid agarose (Invitrogen) fraction, the
hydroxyapatite (Bio-Rad) flow-through or peak fraction, the fraction
after removal of the His6 tag, the peak Superdex 75 or Superdex
200 fraction (GE Healthcare), and the MonoS (GE Healthcare)
flow-through or peak fraction, respectively.
Trang 3The EVH2 domain stimulates RAD51-mediated
homologous pairing
As we reported previously [11], EVL stimulates
homolo-gous pairing by RAD51 We then tested whether the
EVH2 domain also possesses this activity To do this, we
employed the D-loop formation assay, in which an
ssDNA 50-mer and superhelical dsDNA were used as
substrates This combination of DNA substrates
gener-ates D-loops as a product of homologous pairing by
RAD51 (Fig 4A) As shown in Fig 4B (lanes 4–7), EVL
significantly stimulated RAD51-mediated homologous
pairing in the presence of a suboptimal concentration of
RAD51 (50 nm) In contrast, this RAD51 stimulation by
EVL was less obvious in the presence of an optimal
con-centration of RAD51 (340 nm) (Fig 4B, lanes 8–11)
Therefore, we employed the suboptimal RAD51
condi-tions to evaluate the activities of the EVL domains As
shown in Fig 4C (compare lane 4 with lanes 8–10) and
Fig 4D, EVL(222–418) stimulated homologous pairing
by RAD51, although its efficiency was not significant as
compared with that of full-length EVL (Fig 4D) In
con-trast, EVL(1–221) did not stimulate homologous pairing
by RAD51 (Fig 4E,F) These results indicate that the
EVH2 domain is responsible for the stimulation of
RAD51-mediated homologous pairing
Discussion
EVL was originally identified as a member of the
ENA⁄ VASP family, and it reportedly functions in
cytoplasmic actin remodeling [12] In addition to its cytoplasmic function, we previously found that EVL binds to the human recombination protein, RAD51, and stimulates RAD51-mediated homologous pairing and strand exchange reactions in vitro [11] EVL also possesses DNA-binding activity [11], and was found as
a nuclear phosphoprotein by large-scale characteriza-tion of the HeLa cell nuclear fraccharacteriza-tion [13] These facts suggested that EVL may be a novel factor that func-tions in the HRR pathway Human EVL is composed
of 418 amino acids, and contains three distinct domains, EVH1, Pro-rich, and EVH2 [12] To identify the functional domain that is responsible for the recombination-related activity of EVL, we performed a deletion analysis with EVL, which revealed that the EVH2 domain is responsible for the recombination activity of EVL We found that the EVH2 domain: (a) contains the ssDNA-binding and dsDNA-binding activities; (b) binds to RAD51; and (c) stimulates homologous pairing by RAD51
These new findings related to the recombination activities of the EVH2 domain suggest that the ENA⁄ VASP family proteins, which contain the EVH2 domain at their C-terminus, may be involved in DSB repair by homologous recombination in cells In the EVL-knockdown cells, we previously observed a 20–30% reduction in RAD51 foci formation after DSB induction However, the EVL-knockdown cells exhibited slightly increased sensitivity to a DSB-induc-ing agent, mitomycin C (MMC) [11] The weak MMC sensitivity in the EVL-knockdown cells may be due to
Fig 2 DNA-binding activities of EVL(1–221) and EVL(222–418) uX174 ssDNA (20 l M ) and uX174 linear dsDNA (20 l M ) were each incu-bated with the EVL protein at 37 C for 15 min The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining (A) The ssDNA-binding assay (B) The dsDNA-binding assay Lane 1: negative control experi-ments without the protein Lanes 2–4, 5–7, and 8–10: experiexperi-ments with full-length EVL, EVL(1–221), and EVL(222–418), respectively The concentrations of the protein used in the DNA-binding experiments were 0.25 l M (lanes 2, 5, and 8), 0.5 l M (lanes 3, 6, and 9), and 1 l M (lanes 4, 7, and 10) (C) Competitive DNA-binding assay uX174 ssDNA (20 l M ) and uX174 linear dsDNA (20 l M ) were used as substrates in this assay The concentrations of EVL(222–418) were 0.1 l M (lane 2), 0.2 l M (lane 3), 0.4 l M (lane 4), 0.8 l M (lane 5), and 1.2 l M (lane 6) Lane 1 indicates the negative control experiment without the protein.
Trang 4the presence of EVL paralogs, such as the MENA and
VASP proteins Another explanation for the weak
MMC sensitivity in the EVL-knockdown cells is that
the reduced HRR pathway may be complemented by
the nonhomologous DNA end-joining pathway, which
functions as a complementary DSB repair pathway
[14] MENA and VASP are members of the ENA⁄ VASP family [12], and the EVH2 domain, which is responsible for the RAD51-binding, DNA-binding and homologous pairing stimulation, is highly conserved between them (Fig 5) Therefore, the EVH2 domains
of MENA and VASP may also possess these
Fig 3 EVL(222–418) binds to RAD51 (A) His6-tagged EVL, His6-tagged EVL(1–221) and His6-tagged EVL(222–418) were each incubated with RAD51 The RAD51 protein bound to the His6-tagged EVL, EVL(1–221) or EVL(222–418) protein was captured by the Ni 2+ –nitrilotriace-tic acid agarose beads The proteins bound to the Ni 2+ –nitrilotriacetic acid agarose beads were then analyzed by 12% SDS ⁄ PAGE with Coo-massie Brilliant Blue staining Lane 1: molecular mass markers Lanes 2–5: proteins (0.5 lg) used in this assay Lanes 6–8: experiments with His6-tagged EVL, His6-tagged EVL(1–221), and His6-tagged EVL(222–418), respectively Lane 9: control experiment with RAD51 in the absence of His6-tagged EVL, His6-tagged EVL(1–221), and His6-tagged EVL(222–418) (B) Graphic representation of the experiments shown
in (A) The averages of three independent experiments are shown with the standard deviations In this Ni 2+ –nitrilotriacetic acid bead pull-down assay, RAD51 nonspecifically bound to the Ni 2+ –nitrilotriacetic acid beads, and gave a background signal [(A), lane 9] This background signal was reduced in the presence of EVL(1–221), resulting in a negative value in (B) (1–221) (C) RAD51 titration Lane 1: molecular mass markers Lanes 2 and 3: proteins (0.5 lg) used in this assay Lanes 4–7: experiments with RAD51 in the presence of His6-tagged EVL(222– 418) Lanes 8–11: negative control experiments with RAD51 in the absence of His6-tagged EVL(222–418) The RAD51 concentrations were
2 lg (lanes 4 and 8), 4 lg (lanes 5 and 9), 6 lg (lanes 6 and 10), and 8 lg (lanes 7 and 11) (D) Graphic representation of the experiments shown in (C) RAD51 and His6-tagged EVL(222–418), shown in (C), were quantitated The amounts of RAD51 nonspecifically bound to the
Ni 2+ –nitrilotriacetic acid beads were subtracted to determine the amounts of RAD51 bound to EVL(222–418) The vertical axis indicates the EVL(222–418) ⁄ RAD51 molar ratios.
Trang 5nation-related activities, and may complement the
functions of EVL in vivo A comparative study of these
ENA⁄ VASP family proteins is required to clarify the
contributions of these proteins in the HRR pathway
During the cytoplasmic actin-remodeling process,
the ENA⁄ VASP family proteins bind to both the
barbed end and sides of actin filaments [15–18] These
actin-binding activities are conserved in the EVH2
domain [19,20] On the other hand, the EVH1 domain
binds to proteins containing the consensus sequence
D⁄ E FPPPPXD ⁄ E [21–23] The Pro-rich domain also
binds to profilin and proteins containing the SH3 and
WW domains [17,24–26] These facts suggest that the
EVH1, Pro-rich and EVH2 domains all have the
potential to function in protein binding The present
study revealed that the EVH2 domain is the
RAD51-binding domain Minimal RAD51 binding was
observed with EVL(1–221), which contains both the EVH1 and Pro-rich domains, also supporting the con-clusion that the EVH2 domain is the RAD51-binding domain in EVL RAD51 and actin, which both bind ATP, may share a common structural property that is recognized by the EVH2 domain Further studies are required to identify the common sequence or structure that may be recognized by EVH2
Experimental procedures
Protein preparation
The DNA fragments encoding EVL(1–221) and EVL(222– 418) were amplified by PCR, and were cloned in the NdeI site of the pET15b vector (Novagen, Darmstadt, Germany)
In this construct, the His6-tag sequence was fused to the N-terminus of the protein The EVL fragments were expressed
in the Escherichia coli BL21(DE3) strain, which also carried
an expression vector for the minor tRNAs [Codon(+)RP; Stratagene] The cells producing the EVL fragments were resuspended in 20 mm potassium phosphate buffer (pH 8.5), containing 700 mm NaCl, 5 mm 2-mercaptoethanol, 10 mm imidazole, and 10% glycerol, and were disrupted by sonica-tion The cell debris was removed by centrifugation for
A
C
D
pair-ing (A) Schematic representation of the D-loop formation assay Superhelical dsDNA and a 50-mer ssDNA were used as substrates for this assay Asterisks indicate the 32 P-labeled end of the 50-mer ssDNA (B) The D-loop formation assay with EVL Lane 1: control experiment without proteins Lanes 2 and 3: control experiments with EVL and EVL(222–418), respectively, in the absence of RAD51 Lanes 4–7 and 8–11: experiments with a suboptimal RAD51 concentration (50 n M ) and an optimal RAD51 concentration (340 n M ), respectively Lanes 4 and 8: experiments with 50 and
340 n M RAD51, respectively, in the absence of EVL Lanes 5–7 and 9–11: experiments with EVL The EVL concentrations were 0.1 l M (lanes 5 and 9), 0.5 l M (lanes 6 and 10), and 1 l M (lanes 7 and 11) (C) The D-loop formation assay with EVL(222–418) Lane 1: control experiment without proteins Lanes 2 and 3: control experiments with EVL and EVL(222–418), respectively, in the absence of RAD51 Lane 4: experiment with RAD51 in the absence of EVL or EVL(222–418) Lanes 5–7 and 8–10: experiments with EVL and EVL(222–418), respectively, in the presence of RAD51 (50 n M ) The EVL and EVL(222–418) concentrations were 0.1 l M (lanes 5 and 8), 0.5 l M (lanes 6 and 9), and 1 l M (lanes 2, 3, 7, and 10) (D) Graphic representation of the experiments shown in (C) Open and closed circles represent the experiments with EVL and EVL(222–418), respectively The average values of three independent experiments are shown with the standard deviations (E) The D-loop formation assay with EVL(1–221) The same procedure as shown in (C) was used, except that EVL(1–221) was used instead of EVL(222–418) (F) Graphic representation of the experiments shown in (E) Open and closed circles represent the experiments with EVL and EVL(1–221), respectively.
Trang 620 min at 30 000 g, and the lysates were then mixed gently
by the batch method with Ni2+–nitrilotriacetic acid agarose
1 h The beads were washed with 20 mm potassium
phos-phate buffer (pH 8.5), containing 700 mm NaCl, 5 mm
2-mercaptoethanol, 30 mm imidazole, and 10% glycerol, and
were then washed with 20 mm potassium phosphate buffer
(pH 8.5), containing 700 mm NaCl, 5 mm
2-mercaptoetha-nol, 60 mm imidazole, and 10% glycerol The beads were
washed again with 20 mm potassium phosphate buffer (pH
8.5), containing 700 mm NaCl, 5 mm 2-mercaptoethanol,
30 mm imidazole, and 10% glycerol, and were then packed
into an Econo-column (Bio-Rad Laboratories, Hercules,
washed with 38 column volumes of 20 mm potassium
phos-phate buffer (pH 8.5), containing 100 mm NaCl, 5 mm
2-mercaptoethanol, 30 mm imidazole, and 10% glycerol
The His6-tagged EVL fragments were eluted with a 7.5
column volume linear gradient of 30–300 mm imidazole, in
20 mm potassium phosphate buffer (pH 8.5), containing
100 mm NaCl, 5 mm 2-mercaptoethanol, and 10% glycerol
The fractions containing the His6-tagged EVL(1–221)
frag-ment or the His6-tagged EVL(222–418) fragfrag-ment were
diluted with the same volume of 10 mm potassium
phos-phate buffer (pH 8.5), containing 100 mm NaCl, 5 mm
2-mercaptoethanol, and 10% glycerol, and were mixed
gently by the batch method with hydroxyapatite resin (5 mL;
Bio-Rad) at 4C for 1 h The His6-tagged EVL(1–221)
frag-ment did not bind to the hydroxyapatite resin Therefore, the supernatants, which contained the His6-tagged EVL(1– 221) fragment, were collected, and the His6 tag was uncou-pled from the EVL(1–221) portion by digestion with 4 units
of thrombin protease (GE Healthcare Biosciences, Uppsala, Sweden) per milligram of the protein On the other hand, the His6-tagged EVL(222–418) fragment bound to hydroxy-apatite resin Therefore, the resin containing the His6-tagged EVL(222–418) fragment was packed into an Econo-column The resin was further washed with 6 column volumes of
10 mm potassium phosphate buffer (pH 8.5), containing
225 mm NaCl, 5 mm 2-mercaptoethanol, and 10% glycerol, and the His6-tagged EVL(222–418) fragment was then eluted with a 6 column volume linear gradient of 225–
1000 mm NaCl and 10–300 mm potassium phosphate (pH 8.5) The His6 tag was uncoupled from the EVL(222–418) portion by digestion with 4 units of thrombin protease per milligram of the protein The EVL(1–221) and EVL(222– 418) fragments were then immediately dialyzed against
20 mm potassium phosphate buffer (pH 7.5), containing
200 mm NaCl, 5 mm 2-mercaptoethanol, and 10% glycerol,
at 4C After uncoupling of the His6 tag, the EVL(1–221) and EVL(222–418) fragments were further purified by Superdex 75 and Superdex 200 gel filtration column (HiLoad 16⁄ 60 or HiLoad 26 ⁄ 60 prep grade; GE Health-care) chromatography, respectively, followed by MonoS
The peak fractions were diluted with two volumes of 20 mm potassium phosphate buffer (pH 7.5), containing 5 mm 2-mercaptoethanol and 10% glycerol, and were subjected
to MonoS (GE Healthcare) column chromatography The column was washed with 20 column volumes of 20 mm potassium phosphate buffer (pH 7.5), containing 67 mm NaCl, 5 mm 2-mercaptoethanol, and 10% glycerol, and the EVL(222–418) fragment was eluted with a four column vol-ume linear gradient of 67–600 mm NaCl The EVL(1–221) fragment was obtained in the flow-through fraction of the MonoS column, and was concentrated The purified EVL
buf-fer (pH 7.3), containing 100 mm NaCl, 5 mm
Human RAD51 was expressed in E coli cells [27], and was purified by methods described previously [28,29] The con-centrations of the RAD51 and EVL proteins were determined with the Bradford method, using BSA as the standard The concentrations of the EVL(1–221) and EVL(222–418) pro-teins were determined by quantitative SDS⁄ PAGE analysis, using full-length EVL as the standard We then confirmed
generated the same results
Assays for DNA binding
The uX174 circular ssDNA (20 lm) or the uX174 linear dsDNA (20 lm) was mixed with EVL, EVL(1–221) or
B
A
Fig 5 Comparison of amino acid sequences of human ENA ⁄ VASP
family proteins (A) Schematic representation of human EVL,
MENA, and VASP Boxes denoted as EVH1, P-rich and EVH2
repre-sent regions corresponding to the EVH1, Pro-rich and EVH2
domains, respectively (B) The EVH2 sequences were aligned with
CLUSTALW
Trang 7EVL(222–418) in 10 lL of a standard reaction solution,
reaction mixtures were incubated at 37C for 15 min, and
were then analyzed by 0.8% agarose gel electrophoresis in
3 VÆcm)1 for 2 h The bands were visualized by ethidium
bromide staining
The D-loop formation assay
In the D-loop formation assay, superhelical dsDNA
(pB5Sarray DNA) was prepared by a method that prevents
the irreversible denaturation of the dsDNA substrate by
alkaline treatment of the cells harboring the plasmid DNA
The cells were gently lysed using sarkosyl, as described
pre-viously [30] The pB5Sarray DNA contained 11 repeats of a
sea urchin 5S rRNA gene (207 bp fragment) within the
pBlueScript II SK(+) vector The indicated amount of
EVL, EVL(1–221) or EVL(222–418) was incubated in the
7.5), 40 mm NaCl, 0.02 mm EDTA, 0.9 mm
2-mercaptoeth-anol, 5% glycerol, 1 mm MgCl2, 1 mm dithiothreitol, 2 mm
the 32P-labeled 50-mer oligonucleotide (1 lm) was added,
and the samples were further incubated at 37C for 5 min
For the ssDNA substrate used in the D-loop assay, the
following HPLC-purified oligonucleotide was purchased:
50-mer, 5¢-GGA ATT CGG TAT TCC CAG GCG GTC
TCC CAT CCA AGT ACT AAC CGA GCC CT-3¢ (Nihon
Gene Research Laboratory, Sendai, Japan) The reactions
were then initiated by the addition of the pB5Sarray
continued at 37C for 30 min The reactions were stopped
K, and were further incubated at 37C for 15 min After
addition of six-fold loading dye, the deproteinized reaction
products were separated by 1% agarose gel electrophoresis
dried, exposed to an imaging plate, and visualized using an
FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan)
The pull-down assay with Ni2+–nitrilotriacetic
acid beads
Purified His6-tagged EVL, His6-tagged EVL(1–221) or
His6-tagged EVL(221–418) (2 lm) was mixed with RAD51
(2 lm) in 50 lL of binding buffer, composed of 20 mm
Hepes (pH 7.3), containing 95 mm NaCl, 0.1 mm EDTA,
0.042% Triton X-100, 1.7 mm ammonium sulfate, 2 mm
2-mercaptoethanol, 4.2 mm imidazole, and 28% glycerol
After a 30 min incubation at room temperature, a 1.5 lL
aliquot of the Ni2+–nitrilotriacetic acid agarose beads was
added to the reaction mixture, and the RAD51 bound to the His6-tagged EVL fragments was captured by the beads The beads were washed two times with 100 lL of washing
NaCl, 0.1 mm EDTA, 0.05% Triton X-100, 2 mm ammo-nium sulfate, 2 mm 2-mercaptoethanol, 5 mm imidazole, and 30% glycerol The proteins that copelleted with the
Ni2+–nitrilotriacetic acid beads were eluted with a buffer,
3.5 mm 2-mercaptoethanol, 300 mm imidazole, and 21% glycerol The eluted fractions were analyzed by 12% SDS⁄ PAGE with Coomassie Brilliant Blue staining
Acknowledgements
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Sports, Culture, Sci-ence, and Technology, Japan H Kurumizaka is a research fellow in the Waseda Research Institute for Science and Engineering
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