SUPPLEMENTAL ONLINE MATERIALS Supplemental Materials...

SUPPLEMENTAL ONLINE MATERIALS

Supplemental Materials and Methods

Strain and plasmid construction. Strain KLY2962 was generated as described

previously (7). Strains KLY6228 and KLY6231 were generated by crossing KLY2466

and KLY2470, respectively, with KLY2849 {MATα cdc23-1 ade2; a derivative of

RJD1179 (a gift of Ray Deshaies, Caltech, Pasadena, CA)}. To generate strains

expressing NUD1-HA, SLK19-HA, STU2-HA, ASE-HA3, SPC97-HA3, SPC98-HA3, or

TUB4-HA3 under the respective endogenous promoter control, the corresponding loci

were C-terminally tagged with a PCR fragment containing the HA3 epitope using the

method described by Longtine et al. (6). To generate strain KLY6223, strain KLY5401

was mated with strain KLY2675 {MATα URA3::CDC5-HA3; a derivative of YDF13 (a

gift of Leland Johnston, National Institute for Medical Research, London, UK)}. Strain

KLY3098 was generated by crossing strain KLY2962 with strain KLY1548 (α-type

isogenic wild-type). Strain KLY3279 was constructed by integrating a dominant

CDC14TAB6-1 allele (10) at the HIS3 locus.

To generate pKL3266 or pKL3267, a XhoI-StuI DNA fragment bearing either the

NUD1 ORF or the STU2 ORF was cloned into pKL1969 (2) digested with the

corresponding enzymes. Similarly, to construct pKL3205, a StuI fragment bearing SLK19

ORF was inserted into pKL1969 digested with MscI and StuI.

Purification of recombinant proteins from Sf9 cells and kinase assays. To

prepare recombinant proteins, Sf9 cells infected with baculoviruses expressing T7-HA-

Nud1-FLAG, T7-HA-Slk19-FLAG, or T7-HA-Stu2-FLAG were lysed, and then

subjected to pull-down with anti-FLAG M2-agarose (Sigma, St. Louis, MO). Purification

2

of T7-HA-Cdc5-FLAG, T7-HA-cdc5(N209A)-FLAG, GST-Cdc28/His6-Cks1/MBP-

Clb2/GST-Cak1 complex, and GST-cdc28(D145N)/His6-Cks1/MBP-Clb2/GST-Cak1

complex and in vitro kinase assays were carried out as described previously (1). The

resulting samples were then separated by SDS-PAGE as indicated, stained with

Coomassie, and the incorporated 32P was detected by autoradiography.

PBD-binding assays. To prepare mitotic lysates from strain KLY5837 (cdc28-

as1 STU2-HA3), cells were first cultured overnight and treated with nocodazole for 3 h

before harvest. Cells were then lysed with an equal volume of glass beads (Sigma, St.

Louis, MO) in TED buffer {40 mM Tris-Cl (pH 7.5), 0.25 mM EDTA, 1 mM DTT}

supplemented with protease inhibitors. For binding experiment, total cellular lysates were

first diluted with TBSN buffer {20 mM Tris-Cl (pH8.0), 150 mM NaCl, 0.5% NP-40, 5

mM EGTA, 1.5 mM EDTA, 0.5 mM Na3VO4, and 20 mM p-nitrophenyl phosphate

(PNPP)} supplemented with protease inhibitors, and then centrifuged at 15,000 x g for 30

min to clarify heavy cellular materials. The resulting lysates were then incubated with

either bead-bound recombinant GST-fused Plk1 PBD or the corresponding phospho-

pincer H538A K540M (PBD/AM) mutant (3) for 2 h. The bead-associated precipitates

were washed several times with the binding buffer, and then subjected to immunoblotting

analysis.

3

Supplemental References

1. Asano, S., J. E. Park, K. Sakchaisri, L. R. Yu, S. Song, P. Supavilai, T. D.

Veenstra, and K. S. Lee. 2005. Concerted mechanism of Swe1/Wee1 regulation

by multiple kinases in budding yeast. EMBO J. 24:2194-2204.

2. Asano, S., J. E. Park, L. R. Yu, M. Zhou, K. Sakchaisri, C. J. Park, Y. H.

Kang, J. Thorner, T. D. Veenstra, and K. S. Lee. 2006 Direct phosphorylation

and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast. J. Biol.

Chem. 281:27090-27098.

3. Elia, A. E., P. Rellos, L. F. Haire, J. W. Chao, F. J. Ivins, K. Hoepker, D.

Mohammad, L. C. Cantley, S. J. Smerdon, and M. B. Yaffe. 2003. The

molecular basis for phospho-dependent substrate targeting and regulation of Plks

by the polo-box domain. Cell 115:83-95.

4. Gietz, R. D., and A. Sugnino. 1988. New yeast-Escherichia coli shuttle vectors

constructed with in vitro mutagenized yeast genes lacking six-base pair restriction

sites. Gene 74:527-534.

5. Hill, J. E., A. M. Myers, T. J. Koerner, and A. Tzagoloff. 1993. Yeast/E. coli

shuttle vectors with multiple unique restriction sites. Yeast 2:163-167.

6. Longtine, M. S., A. McKenzie, D. J. Demarini, N. G. Shah, A. Wach, A.

Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for

versatile and economical PCR-based gene deletion and modification in

Saccharomyces cerevisiae. Yeast 14:953-961.

4

7. Park, C. J., S. Song, P. R. Lee, W. Shou, R. J. Deshaies, and K. S. Lee. 2003.

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p

regulation and Bfa1p/Bub2p-independent cytokinesis. Genetics 163:21-33.

8. Park, J.-E., C. J. Park, K. Sakchaisri, T. Karpova, S. Asano, J. McNally, Y.

Sunwoo, S.-H. Leem, and K. S. Lee. 2004. Novel functional dissection of the

localization-specific roles of budding yeast polo kinase Cdc5p. Mol. Cell. Biol.

24:9873-9886.

9. Sakchaisri, K., S. Asano, L. R. Yu, M. J. Shulewitz, C. J. Park, J. E. Park, Y.

W. Cho, T. D. Veenstra, J. Thorner, and K. S. Lee. 2004. Coupling

morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. USA. 101:4124-4129.

10. Shou, W., J. H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z. W. Chen,

J. Jang, A. Shevchenko, H. Charbonneau, and R. J. Deshaies. 1999. Exit from

mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14

from nucleolar RENT complex. Cell 97:233-244.

11. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host

strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.

Genetics 122:19-27.

12. Song, S., T. Z. Grenfell, S. Garfield, R. L. Erikson, and K. S. Lee. 2000.

Essential function of the polo box of Cdc5 in subcellular localization and

induction of cytokinetic structures. Mol. Cell. Biol. 20:286-298.

5

Supplemental Figure Legends

Figure S1. A delayed mitotic exit by the cdc23-1 mutation does not suppress the cdc5-

11-associated growth defect. Strains KLY2470 (CDC5), KLY6231 (CDC5 cdc23-1),

KLY2466 (cdc5-11), and KLY6228 (cdc5-11 cdc23-1) were cultured overnight, serially

diluted, spotted on YEP-glucose, and then incubated at the indicated temperature.

Figure S2. Overexpression of TUB4 rescues the microtubule nucleation defect associated

with the cdc5-11 mutation. Strain KLY2466 (cdc5-11) was transformed with the

indicated plasmids. The resulting transformants were treated with nocodazole and then

released into fresh medium. At the indicated time points, cells were fixed and then

subjected to confocal microscopy as described in the Materials and Methods.

Figure S3. Depletion of Cdc5 greatly diminishes proper modification of Nud1, Slk19,

and Stu2. (A-G), To examine whether Cdc5 is required for the modification of indicated

proteins, strain KLY4438 (cdc5∆ + pGAL1-cdc5-1) was C-terminally tagged with a PCR

fragment containing three HA epitopes (HA3) at the respective genomic loci. The

resulting strains KLY5826 (NUD1-HA3) (A), KLY5696 (SLK19-HA3) (B), KLY5828

(STU2-HA3) (C), KLY5707 (ASE1-HA3) (D), KLY5784 (SPC97-HA3) (E), KLY5787

(SPC98-HA3) (F), and KLY5785 (TUB4-HA3) (G) were transformed with either control

YCplac22 vector or YCplac22-CDC5. Cells were cultured overnight in YEP-galactose

medium, arrested with α-factor, and then released to fresh YEP-glucose medium

containing nocodazole. Samples were harvested at the indicated time points and subjected

to immunoblot analyses. Because of the instability of phosphorylated Slk19 in (B), the

6

same membrane was stained with Coomassie (CBB) for loading controls. (H), To

monitor cell cycle progression, an aliquot of the samples harvested was used to determine

the percentages of budded cells by counting >200 cells for each time point after release

from the α-factor block.

Figure S4. Cdc5 is required for proper modification of Nud1, Slk19, and Stu2 in vivo.

(A-C), Strains KLY3098 (cdc5-11) and KLY3279 (cdc5-11 CDC14TAB6-1) were C-

terminally tagged with a three HA-containing DNA fragment at the corresponding

genomic loci. The resulting strains {KLY5855 and KLY5857 (A), KLY5813 and

KLY5816 (B), and KLY5859 and KLY5861 (C)} were arrested in G1 by α-factor

treatment, and then released into fresh YEP-glucose medium at either 23oC or 37oC.

Samples were harvested at the indicated time points for immunoblotting analyses. The

same membranes were then stained with Coomassie (CBB) for loading controls. (D-E),

Aliquots of the cdc5-11 (D) and the cdc5-11 CDC14 TAB6-1 (E) cells were also harvested to

monitor cell cycle progression as in Fig. S2H.

Figure S5. Requirement of Cdc28 activity for proper modification of Nud1, Slk19, and

Stu2 in vivo. (A-C), Strain KLY5401 (cdc28-as1) was C-terminally tagged with a three

HA-containing DNA fragment at the respective genomic loci. The resulting strains

{KLY5849 (A), KLY5820 (B), and KLY5837 (C)} were arrested in G1 by α-factor

treatment, and then released into fresh medium at 30oC. To inhibit Cdc28 activity after

proceeding through G1, 1NM-PP1 (or control DMSO) was added to the cultures 20 min

7

after α-factor release. Total cellular proteins were separated by SDS-PAGE for

immunoblotting analysis with anti-HA antibody. Subsequently, the same membranes

were stained with Coomassie (CBB) for loading controls. Samples harvested at the 120

min time point were counted for budding indices to check cell cycle progression.

Figure S6. The cdc5-11 mutant and several other cdc5 mutants exhibit benomyl-

resistance for growth. (A), Strains KLY2946 (cdc5-1 CDC14 TAB6-1), KLY2950 (cdc5-3

CDC14 TAB6-1), KLY2954 (cdc5-4 CDC14 TAB6-1), KLY2958 (cdc5-7 CDC14 TAB6-1),

KLY2962 (cdc5-11 CDC14 TAB6-1), and KLY2970 (CDC5 CDC14 TAB6-1) were serially

diluted, spotted on YEP-glucose, and incubated at the indicated temperature. The CDC14

TAB6-1 allele was provided to bypass the mitotic exit defect associated with various cdc5

mutations. To test benomyl sensitivity without significantly influencing the cdc5 growth

defect, the benomyl-containing plate was incubated at 30oC. (B), Strains KLY5208

(CDC5), KLY5209 (CDC5∆C-CNM67), KLY5246 (bfa1∆ CDC5∆C-CDC12), and

KLY5210 (CDC5∆C-CNM67 CDC5∆C-CDC12) were spotted as in (A) and then

incubated at 30oC.

Figure S7. Both Cdc5 and Clb2-Cdc28 phosphorylate Nud1, Slk19, and Stu2 in vitro.

(A-C) GST-Cdc28/His6-Cks1/MBP-Clb2/GST-Cak1 and T7-HA-Cdc5-Flag were

reacted with Nud1, Slk19, or Stu2 in the presence of [γ-32P]-ATP at 30oC. Reactions were

terminated by the addition of SDS sample buffer and then separated by SDS-PAGE.

After staining with Coomassie (CBB), the gel was dried and subjected to

autoradiography. Cdc5 W, T7-HA-Cdc5-FLAG; cdc5 N, T7-HA-cdc5(N209A)-FLAG;

8

Cdc28 W, GST-Cdc28/His6-Cks1/MBP-Clb2/GST-Cak1 complex; cdc28 D, GST-

cdc28(D145N)/His6-Cks1/MBP-Clb2/GST-Cak1 complex; Nud1, T7-HA-Nud1-FLAG;

Slk19, T7-HA-Slk19-FLAG; Stu2, T7-HA-Stu2-FLAG. Brackets indicate

phosphorylated signals for Nud1, Slk19, or Stu2, respectively, whereas arrows indicate

autophosphorylated Cdc5. Both wild-type GST-Cdc28 and the kinase-inactive GST-

cdc28(D145N) mutant were phosphorylated by GST-Cak1.

Figure S8. Inhibition of Cdc28 activity results in downregulation of Cdc5. (A-B), Strain

KLY6223 (cdc28-as1 CDC5-HA3) was cultured overnight, arrested in G1 by α-factor

treatment, then released into YEP-glucose containing nocodazole to arrest the cells in

prometaphase. In order to inhibit the mitotic Cdc28 activity at two distinct stages of the

cell cycle, cells were treated with 0.5 µM 1NM-PP1 either 30 min (A) or 70 min (B) after

release from the α-factor block. Total cellular proteins prepared at each time point were

subjected to immunoblotting analyses.

Figure S9. Cdc5-dependent modification of Stu2 requires both Cdc28 activity and the

intact PBD. (A), Mitotic lysates prepared from strain KLY5837 (cdc28-as1 STU2-HA3)

were subjected to pull-down assays with either bead-bound GST-PBD or the

corresponding GST-PBD (H538A K540M) mutant (AM). The precipitates were

immunoblotted with anti-HA antibody. The same membrane was stained with Coomassie

(CBB) for loading controls. Asterisk, a non-specific protein that cross-reacts with anti-

HA antibody. (B), Strain KLY5837 (cdc28-as1 STU2-HA3) was transformed with

pGAL1-CDC5 plasmid (pKL882). The resulting strain was cultured overnight in YEP-

9

raffinose, arrested with α-factor, and then released into YEP-galactose medium

containing nocodazole to express the GAL1 promoter-controlled CDC5. To acutely

inhibit Cdc28 activity without interfering with G1 progression, 1NM-PP1 (or control

DMSO) was added to the cultures 30 min after α-factor release. Total cellular proteins

were prepared at the indicated time points for immunoblot analyses. The same membrane

was stained for loading control. (C), Strain KLY5837 was transformed with either

pGAL1-CDC5 (pKL882), pGAL1-CDC5((W517F V518A L530A) (pKL883), or pGAL1-

CDC5 (N209A) (pKL884). The resulting cells were cultured in YEP-raffinose overnight,

and then shifted to YEP-galactose containing nocodazole for the indicated lengths of

time. Samples were harvested at 120 min after induction and subjected to

immunoblotting analyses.

cdc5-11

cdc5-11

cdc5-11

CDC5

CDC5 cdc23-1

CDC5 cdc23-1

CDC5 cdc23-1

cdc5-11 cdc23-1

23o C

27o C

30o C

CDC5

cdc5-11 cdc23-1

CDC5

cdc5-11 cdc23-1

Park et al., Fig. S1

Park et al., Fig. S2cdc5-11

0 min 9 min 15min

+ pCDC5

+ V

ecto

r+

pTUB4,

2µµµµ

Tub4-HA3

60 min 120 minαααα noc.

+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

Ase1-HA3


+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

BA60 min 120 min

αααα noc.

+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

Nud1-HA3

C

D

Stu2-HA3


+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

Spc97-HA3


+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

Spc98-HA3


+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

Slk19-HA3


+ pC

DC

5

+ ve

cto

r

+ ve

cto

r

+ p C

DC

5

FE

G Hcdc5∆∆∆∆ + pGAL1-cdc5-1

0

20

40

60

100

80

60 90300 120after G1 release (min)

% la

rge-

bu

dd

ed c

ells


CBB

Bn

o t

ag

Cdc5

Nud1-HA3

CB

B


37o C

23o C

37o C

23o C

cdc5-10 cdc5-11 TAB6-1


37o C

23o C

37o C

23o C

no

tag


37o C

23o C

37o C

23o C

Cdc5



37o C

23o C

37o C

23o C

Stu2-HA3

CB

B

C

A


cdc5-11 TAB6-1

60 min23oC 37oC

120 min23oC 37oC23oC

0 min

0

20

40

60

80

100

% c

ells

60 min23oC 37oC

120 min23oC 37oC23oC

0 min

cdc5-11

0

20

40

60

80

100

% c

ells

no

tag


37o C

23o C

37o C

23o C

Cdc5



37o C

23o C

37o C

23o C

CB

B

D

E

Slk19-HA3

B

A


Slk19-HA3

CB

B

90 min 120 min

DM

SO

1NM


0 m

in

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

cdc28-as1

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

0

20

40

60

80

100

% c

ells

90 min 120 min

DM

SO

1NM


0 m

in

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

cdc28-as1

Nud1-HA3

CB

B

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

0

20

40

60

80

100

% c

ells

C

Stu2-HA3

CB

B

90 min 120 min

DM

SO

1NM


0 m

in

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

cdc28-as1

DM

SO

1NM

DM

SO

1NM

DM

SO

1NM

0

20

40

60

80

100

% c

ells

30oC 30oC + 15 µµµµg/ml benomyl

cdc5-1 TAB6-1

cdc5-3 TAB6-1

cdc5-4 TAB6-1

cdc5-7 TAB6-1

cdc5-11 TAB6-1

CDC5 TAB6-1

23oC


B

A

control 5 µµµµg/ml Benomyl 10 µµµµg/ml Benomyl

+ pCDC5

+ pCDC5∆∆∆∆C-CNM67

bfa1∆∆∆∆

+ pCDC5∆∆∆∆C-CNM67+ pCDC5∆∆∆∆C-CDC12

+ pCDC5∆∆∆∆C-CDC12

30oC

cdc5∆∆∆∆

Cdc5

Stu2

: Cdc5W W

+-

NW W D- -- - -

--

+ + + +- -: Cdc28-Clb2: Stu2

W W

+-

NW W D- -- - -

--

+ + + +- -

Autorad CBB

Cdc5

Slk19

: Cdc5W W

+-

NW W D- -- - -

--

+ + + +- -: Cdc28-Clb2: Slk19

W W

+-

NW W D- -- - -

--

+ + + +- -

Autorad CBB

B

A


C

Cdc5

Nud1

Autorad CBB

: Cdc5W W

+-

NW W D- -- - -

--

+ + + +- -: Cdc28-Clb2: Nud1

W W

+-

NW W D- -- - -

--

+ + + +- -

MBP-Clb2

GST-Cak1GST-Cdc28

MBP-Clb2

GST-Cak1GST-Cdc28

MBP-Clb2

GST-Cak1GST-Cdc28

0 20 40 60 80 100

120

140

αααα noc.

DMSO 1NM-PP10 20 40 60 80 10

0

120

140

αααα noc.

0 20 40 60 80 100

120

140

αααα noc.

DMSO 1NM-PP1

0 20 40 60 80 100

120

140

αααα noc.


cdc2

8-as

1 C

DC

5-H

A3

Cdc5-HA3

Cdc28

Cdc5-HA3

Cdc28

A

cdc2

8-as

1 C

DC

5-H

A3

B

A


: pGAL1-CDC5WT

0 min

FA

A

NA

WT

120 min

FA

A

NA

Stu2-HA3

EGFP-HA-Cdc5

0' 120'

YPR

YPG + Noc

αααα-H

Aαααα

-GF

P0

min

40 min

DM

SO

1NM

-PP

1

70 min

DM

SO

1NM

-PP

1

100 minD

MS

O

1NM

-PP

1130 min

DM

SO

1NM

-PP

1

160 min

DM

SO

1NM

-PP

1

160 min

DM

SO

1NM

-PP

1

αααα-H

AC

BB

Stu2-HA3

YPG YPD

cdc28-as1 STU2-HA3 + pGAL1-CDC5

0' 40' 70' 100' 130' 160'

YPR + ααααF

YPG + Noc

1NM-PP1

B

: lysates+ + - -Inp

ut

(4%

)

: GST-PBDWT AM WT AM

Stu2-HA3

Inp

ut

(12%

)

*

GST-PBDαααα

-HA

CB

B

C

Table S1. Strains used in this study

Strain a Genotype Source

KLY1546 a MATa his3-11,15 leu2-3,112 trp1-1 ura3-1 Lab stock

KLY2470 KLY1546 LEU2::TUB1-GFP cdc5∆::KanMX6 (7)

TRP1::CDC5-HA3

KLY2466 KLY1546 LEU2::TUB1-GFP cdc5∆::KanMX6 This study

TRP1::cdc5-11-HA3

KLY6231 KLY2470 cdc23-1 This study

KLY6228 KLY2466 cdc23-1 This study

KLY2946 cdc5∆::KanMX6 LEU2::TUB1-GFP (7)

HIS3::CDC14TAB6-1 TRP1::cdc5-1-HA3







KLY2962 cdc5∆::KanMX6 LEU2::TUB1-GFP This study



HIS3::CDC14TAB6-1 TRP1::CDC5-HA3

KLY5208 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 + (8)

YCplac111-EGFP-CDC5

2

KLY5209 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 + (8)

pRS314-CDC5∆C-CNM67 + YCplac33-GAL1-PLK1

KLY5246 MATa cdc5∆::HphMX4 bfa1∆::his5+ SCC1-HA3::KanMX6 (8)

+ pRS315-CDC5∆C-CDC12 + YCplac33-GAL1-PLK1

KLY5210 MATa cdc5∆::HphMX4 SCCI-HA3::KanMX6 (8)

+ pRS314-CDC5C∆-CNM67 + pRS315-GFP-

CDC5∆C-CDC12 + YCplac33-GAL1-PLK1

KLY4438 MATa cdc5∆::HphMX4 + (9)

YCplac33-GAL1-HA-EGFP-cdc5-1

KLY5826 KLY 4438 HphMX4∆::KanMX6 NUD1-HA3::HphMX4 This study

KLY5696 KLY4438 SLK19-HA3::KanMX6 This study

KLY5828 KLY4438 STU2-HA3::KanMX6 This study

KLY5707 KLY4438 ASE1-HA3::KanMX6 This study

KLY5784 KLY4438 SPC97-HA3::KanMX6 This study

KLY5787 KLY4438 HphMX4∆::KanMX6 SPC98-HA3::HphMX4 This study

KLY5785 KLY4438 TUB4-HA3::KanMX6 This study

KLY3098 MATa cdc5∆::KanMX6 TRP1::cdc5-11 This study

KLY3279 KLY3098 HIS3::CDC14TAB6-1 This study

KLY5855 KLY3098 NUD1-HA3::HphMX4 This study

KLY5857 KLY3279 NUD1-HA3:: HphMX4 This study

KLY5813 KLY3098 SLK19-HA3::HphMX4 This study

KLY5816 KLY3279 SLK19-HA3::HphMX4 This study

KLY5859 KLY3098 STU2-HA3::HphMX4 This study

3

KLY5861 KLY3279 STU2-HA3::HphMX4 This study

KLY5401 MATa cdc28-as1 bar1 (1)

KLY5849 KLY5401 NUD1-HA3::HphMX4 This study

KLY5820 KLY5401 SLK19-HA3::KanMX6 This study

KLY5837 KLY5401 STU2-HA3::KanMX6 This study

KLY6223 MATa cdc28-as1 URA3::CDC5-HA3 This study

a KLY1546 is in W303-1A genetic background.

4

Table S2. Plasmids used in this study.

Name Description Source

YCplac111 CEN, LEU2 (4)

pRS314 CEN, TRP1 (11)

pRS315 CEN, LEU2 (11)

YCplac22 CEN, TRP1 (5)

pSK754 YCplac111, EGFP -CDC5 (8)

pKL 2563 pRS314, CDC5∆-CNM67-GFP (8)

pKL2438 pRS315, EGFP-CDC5∆-CDC12 (8)

pKL321 YCplac22, CDC5 (8)

pKL3266 pBlueBacHis2B, HA-NUD1-Flag This study

pKL3205 pBlueBacHis2B, HA-SLK19-Flag This study

pKL3267 pBlueBacHis2B, HA-STU2-Flag This study

pKL882 YCplac22, GAL1-EGFP2-CDC5∆DB (12)

pKL883 YCplac22, GAL1-EGFP2- (12)

cdc5(W517F V518A L530A)∆DB

pKL884 YCplac22, GAL1-EGFP2-cdc5(N209A)∆DB (12)

SUPPLEMENTAL ONLINE MATERIALS Supplemental Materials...

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