Molecular Cell, Volume 40

Supplemental Information

Temporally and Biochemically Distinct Activities of Exo1 during Meiosis: Double-Strand Break Resection and Resolution of Double Holliday Junctions Kseniya Zakharyevich, Yunmei Ma, Shangming Tang, Patty Yi-Hwa Hwang, Serge Boiteux, and Neil Hunter

Inventory of Supplemental Information

Seven figures and three tables.

Figure S1 is linked to Figure 1. It provides additional information about how DSB-resection

lengths are analyzed and shows DSB-resection profiles over time.

Figure S2 is linked to Figures 2 and 3 and presents independent analyses of meiotic

recombination in wild-type and exo1∆ cells.

Figure S3 presents analysis of noncrossover formation in wild-type and exo1∆ cells.

Figure S4 is linked to Figure 4 and presents analysis of a second nuclease-defective allele of

EXO1, exo1-E150D.

Figure S5 is linked to Figure 5 (and Figures 4 and S6) and compares the spore viability patterns

of wild-type, exo1∆, exo1-D173A and exo1-E150D tetrads.

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Figure S6 is linked to Figure 6 and demonstrates that the crossover defect of exo1∆ is relieved

by additional mutation of the RecQ helicase, SGS1.

Figure S7 is linked to Figure 6 and shows that Exo1 and Mlh3 (the MutLγ complex) function in

the same crossover pathway.

Table S1 presents the raw tetrad data used to calculate genetic map distances in the URA3–

HIS4LEU2 and HIS4LEU2-MAT intervals.

Table S2 presents the raw tetrad data used to calculate genetic map distances for intervals

along chromosome III.

Table S3 lists the full genotypes of strains used.

Supplemental References cite sources of strains and methods used for their construction;

describe the method used for analyzing noncrossover recombinants at HIS4LEU2; and cite the

dHJ dissolution function of the Sgs1-Top3-Rmi1 complex.

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4

Figure S1. Analysis of DSB Component Strands and DSB Resection Over Time.

(A and B) Normalized phosphorimager signal intensities (relative luminescence units) for DSB-

strands detected by 2D native/denaturing Southern analysis using strand-specific probes (see

Figure 1), plotted against the calculated strand-lengths in nucleotides (see Experimental

Procedures). The graphs in the lower panels show the distributions of lengths of the two DSB

component strands.

(C and D) Distributions of meiotic DSB resection lengths over time in wild-type and exo1∆ cells.

Resection lengths were calculated as described in Experimental Procedures.

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Figure S2. Physical Analysis of Meiotic Recombination in Wild-Type and exo1Δ Cells.

(A and B) Quantitative analysis of DSBs, crossovers and meiotic divisions (MI ± MII) for two

independent pairs of wild-type and exo1Δ meiotic cultures. % DNA is percent of total hybridizing

DNA. MI ± MII is cells that have completed either the first or second meiotic divisions as

detected by the number of DAPI-staining bodies. Lower panels show data for DSBs and

crossovers, normalized to the internal maximum.

(C and D) 2D analysis of JMs in wild-type, exo1Δ, ndt80Δ and ndt80Δ exo1Δ cells. JM regions

are magnified in the right-hand panels.

(E and F) Quantification of JMs in wild-type, exo1Δ, ndt80Δ and ndt80Δ exo1Δ cells. % DNA is

percent of total hybridization signal. See Figure 3 for details of JM species analyzed.

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Figure S3. Noncrossover Form at Wild-Type Levels in exo1Δ Cells.

(A) Assay system for the detection of noncrossovers. Open reading frames and diagnostic

restriction sites at the HIS4LEU2 locus are shown. Noncrossovers are detected by virtue of a

BamHI/NgoMIV polymorphism located immediately at the DSB site on the two homologs

(Martini et al., 2006). Following electrophoresis of XhoI-digested DNA, the status of the central

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allele is determined via in-gel digestion with BamHI. The resulting products are resolved in a

second dimension gel and detected by Southern hybridization with Probe 4.

(B) Representative images of native/native 2D noncrossover gels highlighting the DNA species

shown in (A). Crossovers are present in four signals because they can carry either the BamHI or

the NgoMIV allele.

(C) Quantitative analysis of noncrossover formation in two independent meiotic time courses are

shown in panels (i) and (ii). Panel (iii) shows the final noncrossover levels as the means ± S.E.

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Figure S4. Analysis of DSB Resection and Meiotic Recombination in exo1-E150D Cells.

(A) Domains of the Exo1 polypeptide showing conserved N (N-terminal) and I (internal)

nuclease domains, and position of the E150A allele (asterisk).

(B) Comparison of DSB resection profiles in exo1∆ and exo1-E150D cells. The right-hand

panels show representative images of 2D native/denaturing gels hybridized with 5’ bottom

probe.

(C) Genetic analysis of crossing-over in wild-type, exo1Δ and exo1-E150D cells. Cossing-over

in the HIS4LEU2–MAT interval was analyzed by tetrad dissection (see Figure 4 for details of

the interval). Map distances and spore viability are shown. Asterisks indicate significant

differences between map distances p < 0.004 (see Supplementary Table S1). cM,

centiMorgans.

(D) Physical analysis of recombination in wild-type, exo1Δ and exo1-E150D cells. Images of 1D

Southern analysis, and quantitative analysis of DSBs, crossovers and meiotic divisions (MI ±

MII).

(E) Final crossover levels in wild-type, exo1Δ and exo1-E150D cells. Graphs show the averages

of three independent time courses (means ± S.E.).

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Figure S5. Spore Viability Patterns of Wild-Type, exo1Δ, exo1-D173A and exo1-E150D

Tetrads.

The percent of tetrads producing 0,1, 2, 3 or 4 viable spores are shown. See main text for

details.

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Figure S6. Mutation of the RecQ helicase, Sgs1, Relieves the Crossover Defect of exo1∆

Cells

(A) Genetic analysis of crossing-over in exo1Δ, pCLB2-SGS1 and exo1Δ pCLB2-SGS1 tetrads

(Figure 4C shows the intervals analyzed). * significant different by G-test analysis, p < 0.03 (see

Supplementary Table S1).

(B) Spore viability of wild-type, exo1Δ, pCLB2-SGS1 and exo1Δ pCLB2-SGS1 tetrads.

(C) 1D Southern analysis of meiotic recombination.

(D) Quantitative analysis of DSBs, crossovers and meiotic divisions (MI ± MII).

(E) Final crossover levels in wild-type, exo1Δ, pCLB2-SGS1 and exo1Δ pCLB2-SGS1 cells

(analyzed at 24 hrs). Graphs show the averages of three independent time courses (means ±

S.E.).

A protein complex comprising human RecQ DNA helicase, BLM, type-I topoisomerase,

TOPIIIα, and the specificity factor, RMI (RMI1+RMI2) can catalyze the dissociation of dHJs into

two noncrossover duplexes (Singh et al., 2008; Xu et al., 2008). The equivalent complex in

budding yeast, Sgs1-Top3-Rmi1, similarly catalyzes this dHJ “dissolution” reaction in vitro (S.

Kowalczykowski, personal communication), and appears to be responsible for the crossover

defects of mutants that lack pro-crossover factors such as Msh4-Msh5 and Mlh1-Mlh3 (Jessop

et al., 2006; Oh et al., 2007). Specifically, sgs1 mutation alleviates the crossover defects of

msh4, msh5, mlh1 and mlh3 mutants (Jessop et al., 2006; Oh et al., 2007)(data not shown). To

see if the exo1∆ crossover defect is also relieved in the absence of Sgs1, we compared

crossover levels in wild-type, exo1, sgs1 and exo1 sgs1 strains by genetic and physical

methods (Figure 6). To circumvent the synthetic sickness of exo1 sgs1 double mutants (Pan et

al., 2006), the pCLB2-SGS1 mutation was again utilized (described above).

Analysis of tetrads from pCLB2-SGS1 exo1∆ cells shows that map distances are

significantly increased relative to exo1∆ tetrads (P=0.02), and indistinguishable from those

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calculated for wild-type tetrad data (Figure S8). Alleviation of the crossover defect of exo1∆

cells by pCLB2-SGS1 is confirmed by physical analysis of recombination at HIS4LEU2 (Figure

S8C,D,E). The time-course data shown in Figure S8D suggest that restoration may be

incomplete. However, comparison of final crossover levels (at 24 hrs) from three independent

time-courses shows that crossover levels in pCLB2-SGS1 exo1∆ are essentially the same as

those in pCLB2-SGS1 single mutants, but that the pCLB2-SGS1 mutation alone causes a small

reduction in crossing-over relative to wild type (Figure S8E). Thus, genetic and physical assays

suggest that absence of the Sgs1 dHJ-dissolvase activity greatly alleviates the crossover defect

of exo1∆ cells.

If pCLB2-SGS1 completely rescued the spore death of exo1∆ cells, we might expect that

pCLB2-SGS1 exo1∆ tetrads would have the same viability as the pCLB2-SGS1 single mutant,

i.e. 84%. This is clearly not the case as spore viability of pCLB2-SGS1 exo1∆ tetrads is no

different to that of the exo1∆ single mutant (Figure S8B). However, the viability defects of the

two single mutants are clearly not additive, in which case we would expect spore viability of 63%

for pCLB2-SGS1 exo1∆ tetrads. So, at least partial suppression is apparent. Failure of pCLB2-

SGS1 to completely rescue the spore viability of exo1∆ cells could be due to incomplete

suppression of the crossover defect and/or pleiotropic effects and interactions between exo1

and sgs1 mutations.

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Figure S7. Exo1 and Mlh3 Function in the Same Crossover Pathway.

(A) Representative images of 1D Southern analysis of the final crossover levels at the

HIS4LEU2 locus in wild-type, exo1∆ and mlh3∆ single mutants, and the exo1∆ mlh3∆ double

mutant.

(C) Quantitation of final crossover levels at HIS4LEU2. Bars show the averages of three

independent time courses analyzed after 24 hrs (means ± S.E.).

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Table S1. Genetic Distances of the URA3-HIS4LEU2 and

HIS4LEU2-MAT Intervals in Wild-Type, exo1Δ, exo1-D173A, exo1-E150D, pCLB2-SGS1

and pCLB2-SGS1 exo1Δ Strains.

Map distances and standard errors in centiMorgans (cM) were calculated from the distribution of

parental ditype (PD), nonparental ditype (NPD) and tetratype (T) tetrads as described in

Experimental Procedures. ND, not determined.

Interval

URA3–HIS4LEU2 HIS4LEU2–MAT

Genotype PD:N:T cM PD:N:T cM

Wild type 119:2:112 26.61 ± 2.33 109:9:124 36.78 ± 3.68

exo1Δ 164:1:61 14.82 ± 1.94 163:4:62 18.78 ± 2.88

exo1-D173A 115:1:84 22.5 ± 2.23 113:7:82 30.69 ± 3.97

exo1-E150D ND ND 165:6:151 29.04 ± 2.5

pCLB2-SGS1 126:4:114 28.28 ± 2.75 144:6:98 27.02 ± 3.13

pCLB2-SGS1 exo1Δ 132:6:72 25.71 ± 3.63 129:6:82 27.19 ± 3.52

Supplementary Table S2. Genetic Distances in Nine Intervals Along Chromosome III in Wild-Type and exo1-D173A Cells.

Genotype

ADE2:TRP1

TRP1:HIS4

HIS4:LEU2

LEU2:URA3

URA3:LYS2

LYS2:MAT

MAT:THR4

THR4:CUP1

CUP1:kanMX4

Wild type

PD:NPD:T

cM

1567:1:64

2.14±0.3

784:20:79

28.4±1.0

1095:3:473

15.63±0.7

1261:6:353

12.01±0.7

1652:1:0

0.18±0.2

1129:5:511

16.44±0.7

915:18:658

24.07±1.0

756:29:804

30.77±1.1

1337:6:286

9.88±0.6

exo1D173A

PD:NPD:T

cM

315:0:11

1.69±0.5

192:0:125

19.72±1.4

222:1:92

15.56±1.6

243:3:77

14.71±1.9

326:0:0

0

240:1:84

13.85±1.5

203:4:109

21.04±2.2

166:5:141

27.4±2.4

268:0:51

7.99±1.0

Map distances and standard errors in centiMorgans (cM) were calculated from the distribution of parental ditype (PD), nonparental ditype (NPD) and tetratypes (T) tetrads as described in Experimental Procedures. See Figure 5 for details of the chromosome III intervals.

Supplementary Table S3. Strains used in this study.

Strain Genotype

NHY1226*

NHY1296*

NHY1829*

NHY1961*

NHY2242*

NHY2426*

NHY2826*

NHY3235*

NHY3251*

NHY3400*

NHY2328

NHY2468

NHY3638*

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 ndt80Δ::kanMX4/ndt80Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 mlh3Δ::kanMX4/mlh3Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1Δ::kanMX4/exo1Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 pCLB2-SGS1::kanMX4/pCLB2-SGS1::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 ndt80Δ::kanMX4/ndt80Δ::kanMX4

exo1Δ::kanMX4/exo1Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 pCLB2-SGS1::kanMX4/pCLB2-SGS1::kanMX4

exo1Δ::kanMX4/exo1Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 mlh1-E682A/mlh1-E682A

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV) exo1-E150D/exo1-E150D

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1-D173A/exo1-D173A

ade2∆ lys2∆ trp1∆ leu2-r HIS4 CEN3::URA3 MATα thr4-B CUP1

ade2∆ lys2∆ TRP1 LEU2 his4-B CEN3::LYS2 MATa THR4 cup1∆ GIT1::KanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 EXO1-FLAG-kanMX4/EXO1-FLAG-kanMX4

NHY3692

NHY3742

NHY4075*

NHY4116*

NHY4117*

NHY4120*

NHY4135*

ade2∆ lys2∆ TRP1 LEU2 his4-B CEN3::LYS2 MATa THR4 cup1∆ GIT1::KanMX4 exo1-D173A

ade2∆ lys2∆ trp1∆ leu2-r HIS4 CEN3::URA3 MATα thr4-B CUP1 exo-1D173A

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1Δ::kanMX4/exo1Δ::kanMX4

mlh3Δ::kanMX4/mlh3Δ::kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1-247-FLAG-kanMX4/exo1-247-FLAG-kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1-438-FLAG-kanMX4/exo1-438-FLAG-kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3 exo1-504-FLAG-kanMX4/exo1-504-FLAG-kanMX4

MATa/MATα HIS4::LEU2-(BamHI)/his4-X::LEU2-(NgoMIV)—URA3

exo1-FF477AA-FLAG-kanMX4/exo1-FF477AA-FLAG-kanMX4

* These strains are also homozygous for the mutations ura3Δ(sma-pst) and leu2::hisG.

The HIS4LEU2 locus has been described (Hunter and Kleckner, 2001). The exo1Δ mutation was created by replacing gene

coding sequences with the kanMX4 G418-resistance cassette (Wach et al., 1994). The exo1D173A, exo1E150D, pCLB2-SGS1 and

ndt80Δ alleles have been described (Tran et al., 2002) (Allers and Lichten, 2001). The exo1-504, exo1-438, and exo1-247 alleles

were generated by truncating the EXO1 gene with a 3XFLAG-kanMX4 cassette (kindly provided by Dr Akira Shinohara). The full-

length EXO1-FLAG-kanMX4 construct retains full function, as measured by tetrad analysis and DNA physical assays, and was used

as a wild-type control in Figure 6. The exo1-FF447AA allele was constructed by adaptamer-mediated PCR (Reid et al., 2002). The

mlh1-E682A allele was integrated into the MLH1 locus via two-step gene replacement using plasmid pRS306-mlh1-E682 (Dherin et

al., 2009).

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