SUPPLEMENTAL DATA
The Rad52 homologues Rad22 and Rti1 of Schizosaccharomyces pombe are not
essential for meiotic interhomologue recombination, but are required for meiotic
intrachromosomal recombination and mating-type related DNA repair.
Guillaume Octobre*, Alexander Lorenz†‡, Josef Loidl† and Jürg Kohli*§.
*Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland,
and †Department of Chromosome Biology, University of Vienna, Dr Bohr-Gasse 1, A-1030
Vienna, Austria.
‡
Current address: Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, United Kingdom.
§
Corresponding author. Mailing address: Institute of Cell Biology, University of Bern,
Baltzerstrasse 4, CH-3012 Bern, Switzerland. Phone: +41 31 631 46154. Fax: +41 31 631
4616. E-mail: juerg.kohli@izb.unibe.ch
-1-
SUPPLEMENTAL MATERIALS AND METHODS
Spot assays for genotoxin sensitivity: The strains to be assayed were grown to an OD600 of
~0,8 (about 107 cells/ml). The cultures were then serially diluted (10-fold steps) and 10µl of
each dilution was spotted on YEA plates supplemented with the indicated amount of
genotoxins. For each genotoxin-supplemented plate, an unsupplemented YEA control plate
was prepared. The plates were incubated at 30° for 3 days, and then photographed.
Determination of mating efficiency: To determine the mating efficiency of crosses, the
number of cells (C), zygotes (Z), asci (A) and spores (S) in a sample of the crossing material
(see “Material and Methods”) were counted by microscopy. The mating efficiency was
calculated by the following formula: (A+Z+0.25S)/(A+Z+0.25S+0.5C) x 100% (GRISHCHUK
et al. 2004). A minimum of 500 cells were counted in each experiment.
Protein extraction and immunoblotting: Protein extraction was done by trichloroacetic acid
(TCA) precipitation as described previously (FOIANI et al. 1994). For Western blotting,
proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred
to a nitrocellulose membrane (Immobilon-P, Millipore). After overnight incubation in
blocking buffer (PBS + 5% nonfat dry milk), rabbit polyclonal anti-Rad22 or anti-Rti1
antibodies (described in (VAN DEN BOSCH et al. 2002)) were applied at 1:5000 dilution for at
least 1 h at room temperature. The membrane was then washed and incubated at room
temperature with an adequate HRP-conjugated secondary antibody for at least 1 h. The
antibody-bound proteins were detected using the ECL plus Western blotting Detection system
(Amersham Biosciences, USA), and pictures were taken using a LAS-1000 luminescent
image analyser (FUJI, Japan).
-2-
Mitotic whole cell immunostaining experiments: Proteins were localized in
paraformaldehyde-fixed cells following the protocol of (ALFA et al. 1993), except that cell
walls were digested with 0.6 mg/ml Zymolase 100-T (Seikagaku, Japan) for 70 min at 37°.
Polyclonal anti-Rad22 and anti-Rti1 antibodies described above were used at dilutions of
1:500, together with appropriate TRITC-conjugated anti-rabbit secondary antibody. After
staining, cells were mounted in Vectashield mounting medium (Vector Labs, USA) and
examined using a Nikon Eclipse E600 epifluorescence microscope equipped with an Osram
mercury lamp 103W/2. Pictures were taken using a Nikon DXM1200 polychrome digital
camera.
-3-
LITERATURE CITED
ALFA, C., P. FANTES, J. HYAMS, M. MCLEOD and E. WARBRICK, 1993 Experiments with
Fission Yeast. A Laboratory Course Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
FOIANI, M., F. MARINI, D. GAMBA, G. LUCCHINI and P. PLEVANI, 1994 The B subunit of the
DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an
essential function at the initial stage of DNA replication. Mol Cell Biol 14: 923-933.
GRISHCHUK, A. L., R. KRAEHENBUEHL, M. MOLNAR, O. FLECK and J. KOHLI, 2004 Genetic
and cytological characterization of the RecA-homologous proteins Rad51 and Dmc1
of Schizosaccharomyces pombe. Curr Genet 44: 317-328.
OSMAN, F., J. DIXON, A. R. BARR and M. C. WHITBY, 2005 The F-Box DNA helicase Fbh1
prevents Rhp51-dependent recombination without mediator proteins. Mol Cell Biol
25: 8084-8096.
VAN DEN BOSCH, M., K. VREEKEN, J. B. ZONNEVELD, J. A. BRANDSMA, M. LOMBAERTS et al.,
2001 Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces
pombe. Mutat Res 461: 311-323.
VAN DEN BOSCH, M., J. B. ZONNEVELD, K. VREEKEN, F. A. DE VRIES, P. H. LOHMAN et al.,
2002 Differential expression and requirements for Schizosaccharomyces pombe
RAD52 homologs in DNA repair and recombination. Nucleic Acids Res 30: 13161324.
-4-
Table A1: List of the strains used in this study.
Genotypeb
Strain
Matinga
designation
type
1-11
h+
ade1-40
1-26
h-
ade7-50
3-87
h-
leu2-120
3-99
h-
lys4-95
4-150
h+
lys7-2
5-167
h-
ade6-M375
6-216
h+
ade6-469
972
h-
wt
975
h+
wt
51-2009
h+
ade7-152
EPY17
h+
ura4-D18 ura4A-13
EPY5
h-
ura4-D18 ura4A-13
EPY51
h-
smt-0 ura4-D18 ura4A-13
FO1471
h+
rad22::kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4
GO123
h+
rad22::ura4+ ura4-D18 lys4-95
GO124
h+
rad22::ura4+ ura4-D18 lys7-2
GO126
h-
rad22::ura4+ ura4-D18 ade1-40
GO127
h+
rad22::ura4+ ura4-D18 leu1-32 ade7-50
GO156
h+
rti1+::3HA-kanMX6 ade6-M210
GO159
h-
rti1+::3HA-kanMX6 ade6-M216
-5-
GO164
h-
rad22::ura4+ ura4-D18 leu2-120
GO165
h+
rad22::ura4+ ura4-D18 ade6-469
GO170
h-
rti1::LEU2 leu1-32 ade6-469
GO173
h+
rti1::LEU2 leu1-32 ade6-M375
GO175
h-
rti1::LEU2 leu1-32 ade7-152
GO177
h+
rti1::LEU2 leu1-32 ade7-50
GO179
h+
rti1::LEU2 leu1-32 lys4-95
GO181
h-
rti1::LEU2 leu1-32 ade1-40
GO184
h-
rti1::LEU2 leu1-32 ade6-M210
GO187
h+
rti1::LEU2 leu1-32 ade6-M216
GO211
h-
smt-0 rad22::ura4+ ura4-D18 ade7-152
GO214
h-
smt-0 rad22::ura4+ ura4-D18 ade6-M375
GO215
h-
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade7-152
GO218
h-
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade6-M375
GO228
h-
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade1-40
GO229
h+
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade7-50
GO232
h+
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade6-469
GO243
h+
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 lys4-95
GO244
h+
smt0-0 ura4-D18 leu1-32 rec12-Y98F::myc-kanMX6
GO247
h-
GO248
h+
GO260
h-
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 rec12-Y98F::myckanMX6
smt-0 rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 rec12-Y98F::myckanMX6
rad22::kanMX6 rti1::LEU2 ura4-D18 leu1-32 arg3-D4
-6-
a
GO267
h-
smt-0 rad22::kanMX6 rti1::LEU2 ade6-469::ura4+::ade6-M26 ura4D18 leu1-32
GO271
h-
smt-0 rad22::kanMX6 ade6-469::ura4+::ade6-M26 ura4-D18 leu1-32
GO273
h-
smt-0 rti1::LEU2 ade6-469::ura4+::ade6-M26 ura4-D18 leu1-32
GO284
h+
smt-0 rad22::kanMX6 rti1::LEU2 ura4-D18 leu1-32 ade6-D20
GO285
h+
smt-0 rad22::kanMX6 ura4-D18 leu1-32 ade6-D20
GO288
h+
smt-0 rti1::LEU2 ura4-D18 leu1-32 ade6-D20
KLY144
h-
ade6-M210 ura4-D18 rec12-Y98F::myc-kanMX6
MCW1285
h+
rad22::ura4+ ura4-D18 leu1-32 his3-D1 arg3-D4
MCW1489
h+
fbh1::kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4
MCW1553
h+
rad22::ura4+ fbh1::kanMX6 ura4-D18 leu1-32 his3-D1 arg3-D4
MCW1686
h+
rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 his3-D1 arg3-D4
MR61
h+
ade6-D20 ura4-D18
MR62
h-
ade6-469::ura4+::ade6-M26 ura4-D18
RGL10
h+
rti1::LEU2 ura4-D18 leu1-32 ade6-469
RGL13
h+
rad22::ura4+ rti1::LEU2 ura4-D18 leu1-32 ade6-469
RGL6
h+
rad22::ura4+ ura4-D18 leu1-32 ade6-469
All the GO strains were constructed for this study. The RGL strains were a kind gift from A.
Pastink (VAN DEN BOSCH et al. 2001), and the MCW and FO strains from M. C. Whitby
(OSMAN et al. 2005). All other strains were taken from the Berne collection.
b
All the rad22 and rti1 mutants were derived from the MCW1285, MCW1686 and FO1471
strains.
-7-
Table A2: Mating efficiencies in homozygous crosses of rad22 and/or rti1 single and
double mutants.
Average mating
efficiency
(% ± SEM)a
Relative average
mating efficiency (%)b
rad22+ rti1+
(1-11×3-99)
34.7 ± 2.4
100
rad22
(GO123×GO126)
8.2 ± 1.2
23.6
rti1
(GO179×GO181)
30 ± 2.2
86.5
rad22 rti1
(GO228×GO243)
3.7 ± 1.2
10.7
Genotype of the parents
and strain designations
a
The mating efficiency was calculated as indicated in “Supplemental experimental
procedures”. The mean and standard error of the mean (SEM) of 3 independent experiments
are given.
b
Values are relative to the rad22+ level, which was set to 100%.
-8-
Table A3: Spore viability in crosses of rad22 and rti1 single and double mutants.
Genotype of the parents
and strain designations a
Average spore viability
(% ± SEM)a
Relative average
spore viability
(%)b
Number of
independent
experiments
rad22+ rti1+
(1-11×3-99)
86.9 ± 0.7
100
3
38.5 ± 0.9
44.3
3
76.6 ± 5
88.1
3
rad22 rti1
(GO228×GO243)
3.5 ± 1.1
4
3
rad22 rti1
Rec12-Y98F
(GO247×GO248)
14.7 ± 1
16.9
3
Rec12-Y98F
(KLY144×GO244)
18.8 ± 0.5
21.6
3
rad22
(GO123×GO126)
rti1
(GO179×GO181)
a
Means and standard error of the means (SEM) are indicated.
b
Values are relative to the rad22+ level, which was set to 100%.
-9-
Table A4: Intergenic recombination in rad22 and rti1 single and double mutant crosses.
Interval
analysed
ade1-40
lys4-95
leu2-120
lys7-2
a
Average number
Genotype of the Average RF
of colonies
parents and
value ± SEMa analyzed in each
strain
experiment
(%)
designations
Average
genetic
distance
(cM)b
Relative
average
genetic
distance (%)c
rad22+ rti1+
(1-11×3-99)
24.1 ± 1
384
33.4 ± 1.8
100
rad22
(GO123×GO126)
25.1 ± 1.5
384
34.9 ± 2.6
100
rti1
(GO179×GO181)
24.4 ± 1
384
33.6 ± 1.7
100
rad22 rti1
(GO228×GO243)
20.5 ± 0.8
320
26.5 ± 1.4
79.3
rad22+
(3-87×4-150)
10.9 ± 0.7
384
12.2 ± 0.9
100
rad22
(GO124×GO164)
12± 0.4
384
13.7 ± 0.4
100
Recombination frequency (RF) was calculated as the percentage of recombinant colonies
among all colonies analyzed. Mean and standard error of the mean (SEM) were obtained from
three independent crosses.
b
Genetic distance in centiMorgans (cM) was calculated as indicated in Material and Methods.
c
Values are relative to rad22+ level, which is set to 100%.
- 10 -
Table A5: Values of intragenic recombination in rad22 and rti1 single and double
mutants.
Interval
analysed
Range of number of
Genotype of the
prototrophs
Relative average
Average ppm
parents and strain
analyzed
in
each
ppm value
± SEMa
designations
experiment
(%)b
rad22+ rti1+
866 - 1029
100
380 ± 29
(5-167×6-216)
rad22
303 - 406
39.5
150±12
ade6-M375
(GO165×GO214)
ade6-469
rti1
360±17
851 - 1036
94.7
(GO170×GO173)
rad22 rti1
298 - 396
65.8
250±13
(GO218×GO232)
rad22+
973 - 1127
100
610 ± 30
(1-26×51-2009)
rad22
270±15
367 - 431
44.3
(GO127×GO211)
ade7-50
ade7-152
rti1
680±97
911 - 1414
100
(GO175×GO177)
rad22 rti1
460±35
692 - 780
75.4
(GO215×GO229)
a
Prototrophs per million (ppm) were calculated as the number of prototrophic colonies per
million viable spores plated. Spore viability was determined by plating the spores on rich
medium (supplemented YEA). Mean and standard error of the mean (SEM) were obtained
from three independent crosses.
b
Values are relative to rad22+ level, which is set to 100%.
- 11 -
Table A6: Values of intrachromosomal recombination in rad22 and rti1 mutants.
Genotype of the
parents and strain
designations
a
Range of prototrophs Relative average
Average ppm value ±
scored
per experiment
ppm value
SEMa
(%)b
rad22+ rti1+
(MR61×MR62)
80800 ± 2000
2418 - 3228
100
rad22
(GO271×GO285)
13000 ± 2500
188 - 570
16
rti1
(GO273×GO288)
23800 ± 3900
630 - 1111
29
rad22 rti1
(GO267×GO284)
1000 ± 300
1-3
1
Mean and standard error of the mean (SEM) for three independent crosses. Prototrophs per
million (ppm) were calculated as the number of prototrophic colonies per million viable
spores plated. Spore viability was determined by plating the spores on rich medium
(supplemented YEA).
b
Values are relative to wild-type level, which was set to 100%.
- 12 -
SUPPLEMENTAL FIGURE LEGENDS
Figure A1: Comparison of amino acid sequences of Rad52 homologs. The alignment was
realized using the T-COFFEE algorithm. The level of identity between the different regions of
the proteins is indicated by colours, ranging from blue for “different” to red for “identical”.
Gaps in which no alignment could be determined are marked with dashes (-). The line below
the alignment symbolizes the levels of overall amino acid conservation between the proteins,
ranging from highly conserved ones marked with a star (*) to slightly conserved ones marked
with a single dot (.).
Figure A2: Genotoxin sensitivity of rad22 mutant strains. Suppressed (*) and non-suppressed
rad22 deletion strains were cultured and serially diluted. The leftmost spot in each panel
represents 105 cells plated. Strains plated in the rows 5 to 7 were obtained by back-crossing
RGL6, RGL10 and RGL13 (rows 2 to 4) with a wild type strain. rad22 and rad22 rti1
strains display high sensitivity, which can be suppressed by the deletion of the fbh1 gene
(OSMAN et al. 2005).
Figure A3: Immunostaining of whole mitotic cells. White arrows indicate the positions of the
nuclei. (A) wild type, rad22, and rad22 rti1 strains were stained with DAPI (left panels)
and anti-Rad22 antibody (right panels). A nuclear signal for Rad22 was detected. (B) A wildtype strain was stained with DAPI (left panels) and anti-Rti1 antibody (right panels). No
signal could be detected in the nucleus.
Figure A4: Preliminary scheme on the role of Rad22 in avoidance of a lethal lesion possibly
occurring at the mating-type locus. Rad22 is proposed to act at the pausing replication fork at
- 13 -
the smt site of mat1. Single-strand invasion by a Rad51-coated DNA filament into one of the
silent cassettes (mat2 or mat3) on the same or the homologous chromosome, or into mat1 of
the homologous chromosome, is proposed to require Rad22. In the absence of Rad22, a lethal
DNA lesion is proposed to occur. In the absence of functional smt sequence, the imprint
(arrowhead) is not formed, as well as the lesion, resulting in survival of the rad22 spore.
- 14 -