Acharya et al. Supplemental Material
SUPPLEMENTAL METHODS AND MATERIALS
Rad51 protein expression and purification: Protein expression was initially
performed as described by Novagen company literature. Subsequently, to improve
yield we switched to the following procedure, which slightly alters the Novagen protocol
using suggestions by Dr. Joel Ybe at Indiana University. A starter culture of cells
containing the expression construct, beginning with a single colony into 5mls of LBkanamycin (30µg/ml), was grown at 37C for three to five hours or until wisps of bacteria
could be seen and then was stored at 4C overnight. This culture was then used in its
entirety to start a culture of 500ml LB-Kanamycin at room temperature. The cells were
induced, after approximately eight hours of incubation, when the culture had achieved
an OD600 of 0.2-0.4, by adding IPTG to a final concentration of 1mM. After induction the
cells were allowed to grow overnight at room temperature until the culture was
saturated. Induced cells were pelleted and stored at -20C or their expressed protein
was extracted and purified immediately, with no differences in protein yield.
The cell pellets were lysed using the Bugbuster (Novagen) cell lysis reagent, for
15 to 20 minutes with shaking, using 5ml/g of cell pellet, along with 5l of benzonase
nuclease and 50l protease inhibitor cocktail III (Novagen/Calbiochem) per gram of cell
pellet. The lysate was then spun at 16,000g for 20 minutes, to separate soluble and
insoluble fractions. The Rad51 protein was located in the soluble fraction, which was
then used in further purification steps.
Rad51 protein products were purified using cellulose Ni+2 columns under
vacuum, following the Novagen protocol exactly. Purified protein was used for
1
immunization of animals, for construction of columns needed to purify immune sera, and
for later antibody testing. Approximately 0.5mg purified Rad51 was loaded onto an 8%
SDS-acryamide gel, and run into the gel for 2 hours. The gel was then stained with
Coomassie brilliant blue, and the Rad51 band was excised from the gel, and used as
antigen for the anti-Rad51 antbody.
Affinity purification column construction: In order to purify the immune sera
received from Cocalico, we constructed affinity purification columns that were able to
specifically bind the anti-Rad51 antibody or to bind antibodies raised against the vectorencoded protein tags, by constructing an affinity purification column with Rad51 bound
to it and a second column with another protein, unrelated to Rad51, expressed from the
pET-30 vector. The following is derived from Bio-Rad literature, with modification; both
proteins were bound to the columns in the same manner as in the Rad51 column
construction described here. Rad51 was concentrated to approximately 1mg/ml, by
using protein spin columns (Pall Life Sciences Nanosep columns or Millipore Microcon
columns) and polyethylene glycol chips (MW 15,000) to extract water from protein in
solution in dialysis tubing (MW cut off 3500, prepared as in ref. (SAMBROOK 2001)). The
concentrated Rad51 was then dialyzed into MOPS buffer (50mM MOPS, 50mM NaCl,
0.1mM EDTA, 0.1mM EGTA, and 0.1%SDS pH 7.2). Five mg of Rad51 was prepared
in 5ml of MOPS buffer, and a 20l sample of the protein was retained. Using a fretted
funnel, 2ml of a 1:1 slurry of Affigel-15 (Bio-Rad) to isopropanol, which was stored at 80C, was washed with 5ml ice cold 100% ethanol, 5ml ice cold 50% ethanol, 5ml ice
cold water, and 5ml room temperature MOPS buffer, all within 15 minutes, keeping the
Affigel-15 wet at all times. Immediately after the last wash, the Affigel-15 was scooped
2
into the Rad51 solution and mixed gently at room temperature for one hour, allowing the
reactive groups on the Affigel-15 to covalently bind Rad51 protein. After binding, the gel
was transferred to a column (Kontes glass co. cat# 420400) and allowed to drain. The
flow-through was saved and used to represent the sample after binding. To remove
excess unbound Rad51, the column was washed three times with 5ml of MOPS buffer
and then three times with 10ml of MOPS buffer. The remaining unbound active sites of
the Affigel-15 were blocked by washing with 5ml of 1M Tris pH 8.0 for one hour. The
column was then exposed to 5ml of each of the solutions used during antibody affinity
purification, to ensure that any reactivity between the column and antibody purification
solutions occurred before antibody purification to prevent a decrease in antibody yield.
These solutions included TBST [0.5M NaCl, 20mM Tris HCl pH 7.4 (TBS) + 0.2 % Triton
X-100], glycine elution buffer (0.15M Glycine-HCl pH2.0), guanidine elution buffer (6M
Guanidine HCl in TBS), and TBS. The column was then stored under TBS at 4C until
used.
Affinity purification of immune sera: We performed the following protocol twice for
the purification of anti-Rad51 antibody, once to bind antibodies raised against the
vector-encoded tags to remove them from the sera, and a second time to purify the antiRad51 antibody. This protocol is derived from that of the Walczak lab at Indiana
University (W ALCZAK et al. 1996). First, a column prepared with another protein
unrelated to Rad51, expressed from the pET-30 vector, was washed with 5ml of low salt
TBS (0.15M NaCl, 20mM Tris pH7.4). A 5ml volume of immune serum was then diluted
1:1 with TBS and passed over the column five times. The sera flow-through was then
3
passed over the Rad51 column five times. Final sera flow-through from the Rad51
column was saved at 4C.
The Rad51 column was then washed with 25ml of TBS + 0.2% Triton X-100 and
then 25 ml of TBS to remove antibodies and other proteins from the sera that adhere
non-specifically. Rad51 antibody was then eluted in two stages. First, a low pH elution
was performed using glycine elution buffer. After the TBS wash the column was
allowed to run until almost dry. Fifteen ml of glycine elution buffer were pipetted on to
the column 1ml at a time. Each ml was eluted into a 1ml fraction. As each fraction was
eluted off the column the receiving tubes were prepared with 100l of 2M Tris-HCl pH
8.5 to neutralize the acidic elution buffer. The column itself was then neutralized by
washing with 25ml of TBS or with TBS until the pH was 7.4. A second round of elution
was then performed to elute antibodies that did not disassociate from the protein on the
column under low pH. This second elution used 15 ml of guanidine elution buffer, done
in the same manner as the glycine elution except without any neutralization solution in
the receiving tubes. After the last elution, the column was regenerated by washing with
25ml of TBS. The elution fractions were tested by western blot for presence of
antibody. Typically, the majority of antibody was recovered in the glycine fractions and
the guanidine fractions contained too little antibody to be recovered. Fractions
containing anti-Rad51 antibody were combined, concentrated and dialyzed into PBS
(137mM NaCl, 2.7 mM KCl, and 10mM Na2HPO4) by standard procedures (SAMBROOK
2001). The column used to capture antibodies generated against tags encoded by the
pET-30 vector was regenerated by performing the same elution procedure as used for
the Rad51 column, with the elution factions being discarded.
4
Characterization of C. cinereus anti-Rad51: In order to investigate meiotic
recombination we purified an anti-Rad51 antibody, derived from rabbits immunized with
a full-length Rad51 fusion protein. Western blot analysis identified a band at 45kD,
similar to the predicted protein size of Rad51 at 37kD (Figure S2A). Bands observed at
80 and 120kD probably represent poorly disassociated multimers of Rad51 and bands
seen at 20 kD are secondary antibody specific bands (data not shown). Comparison
with commercially available anti-Human Rad51 antibody showed that the two antibodies
recognize bands of the same size (Figure S2B). The Rad51 band in C. cinereus
extracts runs as a doublet when stained with either anti-C. cinereus Rad51 or antiHuman Rad51, which likely results from partial phosporylation of the protein (CHEN et al.
1999; LI et al. 2002; SLUPIANEK et al. 2001; YUAN et al. 1998).
Because Rad51 is a highly conserved protein, we also asked if our anti-Rad51
antibody would recognize the meiosis-specific Rad51 paralog, Dmc1. We obtained as a
gift from the Sakaguchi lab an expression construct for C. cinereus Dmc1 in the pET-28
vector (NARA et al. 2001), and used our standard purification techniques to obtain
Rad51 and Dmc1 proteins, which we analyzed on a western blot probed anti-Rad51
antibody. Rad51 was detected, but Dmc1 was not (Fig. S2C), indicating that the antiRad51 antibody is specific to Rad51 and does not cross react with Dmc1 at this protein
concentration.
In a further test of the specificity of the anti-Rad51 antibody, we found that the
foci observed on wild-type chromosome spreads by immunofluorescence microscopy
could be competed away by a ten-fold molar excess of purified Rad51 protein (data not
shown).
5
Nucleolar staining of Rad51: Diffuse nucleolar staining of Rad51 was observed with
both anti-Rad51 antibody and when secondary antibody was used alone, and was not
counted as nuclear foci. However, punctate foci in the nucleolus were observed
specifically with anti-Rad51 antibody, as was seen previously by others (BARLOW et al.
1997; FRANKLIN et al. 1999) and may represent either DNA repair events or a reservoir
of Rad51 protein. All of the immunofluorescence staining results described here were
acquired by counting the total number of foci on chromosome spreads, i.e., counting
foci present on non-nucleolar chromatin and in the nucleolus. The numbers of Rad51
foci observed in the nucleolus and in the rest of the nucleus were counted for one slide
each of wild type, spo11-1, rad50-1, and rad50-8 at both K+1 and K+4 (N=20-35 per
slide). In all instances the mean proportion of foci observed in the nucleolus was
between 15% and 25% of the total number of foci observed in the bulk chromatin and in
the nucleolus combined. From this we concluded that there were no differences among
our strains regarding the proportion of nucleolar staining, and thus that counting the
total number of Rad51 foci gave biologically meaningful comparisons among the strains.
Rad51 foci in spo11-1: To examine the capacity to form Rad51 foci in spo11-1, we
irradiated spo11-1 caps at K+1 with 60 Krad of gamma radiation (Figure S4). We then
made K+2 or K+4 chromosome spreads using the irradiated cap tissue and stained
them with anti-Rad51 antibody. We observed a dramatic increase in the number of foci,
which approached or exceeded wild-type levels of Rad51 staining during meiosis.
Thus, in spo11-1 Rad51 foci are induced in response to DNA damage. This indicates
that the anti-Rad51 antibody is able to recognize foci formed after irradiation, and that
6
the defect in spo11-1 is in DSB formation, as previously inferred (CELERIN et al. 2000),
rather than some other aspect of Rad51 focus formation.
The observation of a low level of Rad51 foci in spo11-1 could be staining
background, (i.e., a technical artifact) or might reflect recombination or repair events
that are not Spo11 dependent. In order to distinguish between technical and biological
backgrounds, we examined staining of meiotic chromosome spreads using a nonspecific antibody (IgG from rabbit serum), at a concentration of 5g/ml, the same
concentration used for the anti-Rad51 antibody (Figure S4F). We stained one slide
each of meiotic chromosome spreads from a spo11-1 mushroom at K+1 and K+2. We
found that at K+1 and K+2 the number of foci observed by staining spo11-1 with the
non-specific antibody (mean = 294 at K+1, 173 at K+2) was significantly less than the
number of foci observed staining spo11-1 with anti-Rad51 (mean = 435 at K+1, 444
at K+2; p<0.001 for both K+1 and K+2).
We also examined wild type by staining with the same non-specific antibody,
staining one slide each from meiotic chromosome spreads made at K+1, K+2, K+3, and
K+4. In wild type the number of foci observed for the non-specific antibody, at all time
points examined, was less than or equal to the non-specific antibody staining observed
for spo11-1 at K+2. Because some foci were observed when staining with non-specific
antibody in both wild type and spo11-1, a proportion of Rad51 foci observed are likely
background foci and an artifact of the staining procedure. The Rad51 foci not explained
as background might be derived from a low level of Spo11-independent recombination,
DNA repair events, or a reservoir of inactive Rad51 molecules.
7
Image analysis: Foci were counted using the Metamorph imaging system (Molecular
Devices) as follows. The FITC image was first processed using the “flatten background”
command, producing a new image in which diffuse staining was removed. A
background area of this new image, which did not correspond to an area of chromatin
as seen in the DAPI image or spurious background in the FITC image, was selected.
This area selected was used in the “use region for background” command, which
produced a new image that subtracted the average pixel intensity in the selected region,
thereby dramatically reducing camera noise. An inclusion threshold was selected,
which was set between the maximum pixel brightness and a setting just above the point
at which background camera noise was selected. This selected a threshold at which all
of the foci, but none of the background, was selected. The number of objects was
counted using the “Integrated Morphometry Analysis” (IMA) tool of Metamorph
(Molecular Devices). In order to ensure that foci contacting each other were not
counted as one object, the number of foci was counted using the standard area count
tool in IMA, with the standard area configured at 35. This tool uses a standard value
representing a focus size to calculate what the number of distinct objects would be in a
large object that is typically encountered when many foci overlap. Measurements for
each image were logged to Excel (Microsoft) for further analysis.
Image display: In the Metamorph imaging system (Molecular Devices) images can be
acquired with user-defined exposure times, or with computer controlled exposure times.
In addition, each image can be displayed such that the range of pixel brightness
displayed corresponds to the image acquired or corresponds to a user-defined range.
All DAPI images were acquired using the auto exposure command and displayed using
8
the full range of pixel brightness according to each image. FITC images were acquired
using a constant exposure time of 7.5 seconds, and are displayed using the full range of
pixel brightness for each image. Images from different strains and time points, and
even within the same strains and time points, had variable maximum and minimum pixel
brightness levels between images. If images were displayed on a uniform scale of pixel
intensity, then, because of their inherent variability, the images would not represent the
foci accurately (Figure S3).
9
LITERATURE CITED
BARLOW , A. L., F. E. BENSON, S. C. W EST and M. A. HULTEN, 1997 Distribution of the
Rad51 recombinase in human and mouse spermatocytes. Embo J 16: 52075215.
CELERIN, M., S. T. MERINO, J. E. STONE, A. M. MENZIE and M. E. ZOLAN, 2000 Multiple
roles of Spo11 in meiotic chromosome behavior. Embo J 19: 2739-2750.
CHEN, G., S. S. YUAN, W. LIU, Y. XU, K. TRUJILLO et al., 1999 Radiation-induced
assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl.
J Biol Chem 274: 12748-12752.
FRANKLIN, A. E., J. MCELVER, I. SUNJEVARIC, R. ROTHSTEIN, B. BOWEN et al., 1999 Threedimensional microscopy of the Rad51 recombination protein during meiotic
prophase. Plant Cell 11: 809-824.
LI, Y., H. SHIMIZU, S. L. XIANG, Y. MARU, N. TAKAO et al., 2002 Arg tyrosine kinase is
involved in homologous recombinational DNA repair. Biochem Biophys Res
Commun 299: 697-702.
NARA, T., F. HAMADA, S. NAMEKAWA and K. SAKAGUCHI, 2001 Strand exchange reaction
in vitro and DNA-dependent ATPase activity of recombinant LIM15/DMC1 and
RAD51 proteins from Coprinus cinereus. Biochem Biophys Res Commun 285:
92-97.
SAMBROOK, J. A. R., D., 2001 Molecular Cloning A Laboratory Manual. Cold Spring
Harbor Laboratory Press, NY.
SEITZ, L. C., K. TANG, W. J. CUMMINGS and M. E. ZOLAN, 1996 The rad9 gene of Coprinus
cinereus encodes a proline-rich protein required for meiotic chromosome
condensation and synapsis. Genetics 142: 1105-1117.
SLUPIANEK, A., C. SCHMUTTE, G. TOMBLINE, M. NIEBOROWSKA-SKORSKA, G. HOSER et al.,
2001 BCR/ABL regulates mammalian RecA homologs, resulting in drug
resistance. Mol Cell 8: 795-806.
WALCZAK, C. E., T. J. MITCHISON and A. DESAI, 1996 XKCM1: a Xenopus kinesin-related
protein that regulates microtubule dynamics during mitotic spindle assembly. Cell
84: 37-47.
YUAN, Z. M., Y. HUANG, T. ISHIKO, S. NAKADA, T. UTSUGISAWA et al., 1998 Regulation of
Rad51 function by c-Abl in response to DNA damage. J Biol Chem 273: 37993802.
10
Table S1. rad50 intronsa,b
intron
bp
5’ splice junction
internal sequence
3’ splice junction
1
50
AGgtgtgcaccgcgtctggtctgcaggaattaacgagctgaagaaattccagAG
2
51
AAgtctgtcgtctctttccatctctgtgcaacaaagctgacagtttctaacagAT
3
50
TGgtaagatttatttccatcttctttgaagaactacgttgataggttgatagGC
AGgtgggcctgttcatccaaacttccagagtgacggctcaacttcattcagAATCAAAGc
4
49
5
53
AGgtttgtcatttactcgctcaggatacgttgaggccttaactcggactctgtagTT
TGgtaggacttacgttcttctcgtctgacattatgctcaaattgttctcagAG
6
49
7
48
ATgtgagcgacctcgtacttctggttcttcatcactgacactgctggtagCA
8
54
CGgtaagtgtttccgtcgtcgctacgctgaagcggggttctacgtcttttgtatagTC
9
56
AAgtatggtcatattaccttgttttgttcactgccaggctaaaattcttgaattaaagAG
10
50
AGgtaggtgttgccttctgtccgatatgggcaccgccgctgacattacacagTG
11
53
ACgtaagtctcgctcatttcatattccagtacatatacagctaacgtctggtcagTT
12
48
CGgttagtgggatgcatatatctttttcgaggtaaaactgactcgaacagCT
13
53
CTgtaactattcactcactcctttggctacttgggcgatactgacatcccgatagAC
14
53
AGgtgcgtttttccccattcgttagcaaatggtgatgtgaagccactatgtgtagGA
15
53
CGgtgtgcttcttatcacttggttaaaaagcttccccgctgaatggaaattctagGT
16
50
CGgtgagtggtacccgtcgatcctaaggcctcagctcttaacttctcccaagAC
17
63
AAgtaagtcaccccttttggccatagtgaatacacagtgacggcttgcgctcgctgcgatgaaagAT
18
55
AAgtaagtccttcccaatatggtgcacacgtgtccgttctcaactatgtcaattaagCG
19
49
ATgtttgtgaccgccactcctgctagaattcccgcgcttacaccttttcagAT
20
71
GAgtcagtactttctatacccattccgctcaccatcggtcaggtgttgactccgcaactttcacgttctttagGT
21
51
GAgtacgttcttcttccctatctcgttcccaaatattctaatccaattcccagCA
22
52
TGgtgggtgattaccagcaatctggcttccaattgttaactcaaattatctcagGC
average
52.7
aExon sequences are upper case capital letters and intron sequences are in lower case
letters. Boldface type indicates 5’ and 3’ splice junctions (SEITZ et al. 1996)
bUnderlined
cAdditional
bases denote branch point consensus sequences (SEITZ et al. 1996).
sequence for exon 5 is given; italics denote the splice junction site used in
rad50-1.
11
SUPPLEMENTAL FIGURE LEGENDS
Figure S1.-- Splice sites used in rad50-4. The end of wild-type intron 20 is shown in
lower case letters. Boldface type indicates splice acceptor sites.
Figure S2. -- Western blot analysis of the C. cinereus anti-Rad51 antibody: (A)
Nuclei prepared from mushroom caps were lysed in sample buffer containing 8M urea,
and separated using SDS-PAGE with a 4 to 15% gradient gel. The gel was blotted onto
nitrocellulose and probed with C. cinereus anti-Rad51 at 1g/ml. (B) Extracts prepared
as in (A), except that the sample buffer did not contain urea, were probed with Human
anti-Rad51 at a 1:1000 dilution of undiluted serum. (C) C. cinereus anti-Rad51 was
used at a concentration of 1g/ml to probe a blot with 2ng of Rad51 fusion protein and
2ng of Dmc1 fusion protein. Arrowheads for all panels indicate the position of Rad51
monomer.
Figure S3. -- Image display.-- Wild type (A-C) and spo11-1 (D-F) images are shown.
(A, D) DAPI images are acquired using the “auto exposure” command in Metamorph
(Molecular devices). (B,C,E,F) FITC images from all strains were taken using the same
exposure time (7.5 seconds). Images in (B) and (E) are displayed using the full range
of pixel brightness for each image. Images in (C) and (F) are identical to (B) and (E)
except that they are displayed with a range of pixel brightness corresponding to the
combined range of the wild type and spo11-1 FITC images. Images in Figure 4 were
displayed use the display preferences shown in (B) and (E). Scale bars represent 2 m.
12
Figure S4. -- Irradiation induces Rad51 foci in spo11-1: (A) spo11-1 at K+4. (B)
spo11-1 at K+4 after 60Krad of gamma radiation at K+1. (C) spo11-1 stained
with non-specific antibody at K+2. Scale bars represent 2m for all images
13