1

Supplementary Material Contents: Arlow, Scott, Wagenseller, and Gammie Supplementary Table 1: Missense Substitutions Causing Decreased Msh2 Levels ....................... 2

Supplementary Table 2. Original Sources MSH2 Missense Alleles ............................................... 3

Supplemental Table 3: Strains used in this study ........................................................................... 4

Supplementary Table 4: Plasmids expressing MSH2 alleles and vector controls .......................... 6

Supplementary Figure 1: Stability residues cluster to four regions on hMutSα ............................. 8

Supplementary Data: Cisplatin Sensitivity ..................................................................................... 9

Supplementary Methods ............................................................................................................... 10

Mismatch Repair ViabilityAssay .............................................................................................. 10

Turnover Assays ....................................................................................................................... 11

Growth Rate Correction in Turnover Experiments ................................................................... 11

References ..................................................................................................................................... 13

2

Supplementary Table 1: Missense Substitutions Causing Decreased Msh2 Levels

Cluster (Domains)

Yeast Position Human Position Substitution* Detection of variant in

tumor† Inner core

(1/2) E194 E198 G n/d C195 C199 R/Y - L183 L187 P -

Central Core

(3/4/5) C345 C333 R/Y/F -‡ G350 G338 R n/d T347 T335 I n/d A618 A600 V - D621 D603 G/N -

Upper Core

(3/5) G711 G692 D/R n/d C716 C697 F/R -§ P640 P622 L/T n/d

R371 R359 S -

DNA binding (4/3/5)

L521 L503 P n/d D524 D506 Y n/d R542 R524 P/L n/d

*Original sources in Supplementary Table 2 † Immunohistochemistry of tumor samples (-) indicates no detectable hMsh2 protein; n/d, not done ‡ C to F variant examined § C to Y variant examined

3

Supplementary Table 2. Original Sources MSH2 Missense Alleles Yeast Human Original Source L183P L187P (1) E194G E198G (2) C195R C199R (3, 4) C345F C333F (Lenka Foretová et al.) C345R C333R (de la Chapelle et al.) C345Y C333Y (5, 6) T347I T335I (7) G350R G338R (8) R371S R359S (9) L521P L503P (Myriad Genetic Laboratories) D524Y D506Y (10) R542L R524L (11) R542P R524P (12) A618V A600V (13) D621G D603G (14) D621N D603N (15) P640L P622L (16) P640T P622T (17) G711D (18) G711R G692R (19) C716F C697F (20) C716R C697R (14, 19, 21)

4

Supplemental Table 3: Strains used in this study Name Genotype Source 111-A-8 MATα san1Δ::kanMX4 lys2Δ0 ura3Δ0 leu2Δ0 his3Δ1 (22) 131-B -1 MATα msh2Δ::kanMX4 ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0 (22) 20-A -8 MATa pdr5Δ::KanMX4 met15Δ0 ura3Δ0 leu2Δ0 his3Δ1 (22) 30-D -5 MATα pib1Δ::KanMX4 lys2Δ0 ura3Δ0 leu2Δ0 his3Δ1 (22) 45-F -7 MATα doa10Δ::KanMX4 lys2Δ0 ura3Δ0 leu2Δ0 his3Δ1 (22) AGY75* MATα msh2Δ::LEU2 ade2-1 trp1-1 ura3-1 leu2-3,112

his3-11,15 This laboratory

AGY227* MATα ade2-1 trp1-1 ura3-1 leu2-3,112 his3-11,15 This laboratory AGY1057 MATa pol3-01 msh2Δ::LEU2 leu2-3,112 his3Δ200 trp1-1

ura3-1 [MSH2-MYC:: KanMX6 URA3 CEN/ARS] This laboratory

BY4742 MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0 (22) MY11331 MATa msh2Δ::kanMX4 erg6Δ::NatMX ura3Δ0 leu2Δ0

his3Δ1 met15Δ0 This laboratory

MY11333 MATa leu2Δ0 ura3Δ0 met15Δ0 his3Δ1 erg6Δ::NatMX This laboratory MY11719£ MATα pre1-1 pre2-2 ura3Δ5 his3-11,15 leu2-3,112 (23) MY12064* MATa hom3-10 msh2Δ::URA3 leu2-3,112 ade2-1 trp1-1

ura3-1 his3-11,15 This laboratory

MY12078* MATa his3-11,15 trp1-1 ura3-1 leu2-3,112 hom3-10 pdr5Δ::KanMX4 msh2Δ::LEU2 erg6Δ::TRP1

This laboratory

MY12336* MATa hom3-10 msh2Δ::URA3 leu2-3,112 ade2-1 trp1-1 ura3-1 his3-11,15 san1Δ::NatMX

This laboratory

MY12516 MATα pre1-1 pre2-2 ura3Δ5 his3-11,15 leu2-3,112 san1∆::NatMX

This Laboratory

MY9691* MATα msh2Δ::URA3 his3-11,15 ade2-1 ura3-1 trp1-1 This laboratory PJ69-4A MATa trp1-901 leu2-3,112 ura3-52 his3Δ200 gal4Δ

gal80Δ GAL2-ADE2 LYS2::GAL1-HIS3 met2::GAL7-lacZ

(24)

PJ69-4α MATα trp1-901 leu2-3,112 ura3-52 his3Δ200 gal4Δ gal80Δ GAL2-ADE2 LYS2::GAL1-HIS3 met2::GAL7-lacZ

(24)

* W303 strains. Confirmed to be wild-type at the RAD5 locus by PCR and at the CAN1 locus by canavanine resistance assays. £Strain obtained from Kiran Madura, UMDNJ-Robert Wood Johnson Medical School

Kanamycin marked gene deletions were amplified from strains obtained from the Yeast Deletion

Consortium (22) and engineered into W303 strains using single-step PCR-mediated gene

disruption (25, 26). Molecular confirmation of proper gene replacement was achieved by PCR

amplification using primers ~200bp up and downstream of the locus in question. Contingent with

5

survival, deletions in ERG6 were made in a TRP1 background (27). Because erg6∆ strains have

a reduced capacity for chemical transformation, electroporation was employed as described

previously (28). All other strains were transformed using the lithium acetate protocol (29).

6

Supplementary Table 4: Plasmids expressing MSH2 alleles and vector controls Plasmid Number

Plasmid Name Relevant Markers Source

pRS413 HIS3 CEN6 ARSH4 ampr (30) pAG17 pMSH2 MSH2 HIS3 CEN6 ARSH4 ampr (18) pAG486 pMSH2-L183P msh2-L183P HIS3 CEN6 ARSH4 ampr This work pAG375 pMSH2-E194G msh2-E194G HIS3 CEN6 ARSH4 ampr (18) pAG207 pMSH2-C195R msh2-C195R HIS3 CEN6 ARSH4 ampr (18) pAG495 pMSH2-C195Y msh2-C195Y HIS3 CEN6 ARSH4 ampr This work pAG507 pMSH2-C345F msh2-C345F HIS3 CEN6 ARSH4 ampr This work pAG208 pMSH2-C345R msh2-C345R HIS3 CEN6 ARSH4 ampr (18) pAG209 pMSH2-C345Y msh2-C345Y HIS3 CEN6 ARSH4 ampr (18) pAG417 pMSH2-T347I msh2-T347I HIS3 CEN6 ARSH4 ampr (18) pAG418 pMSH2-G350R msh2-G350R HIS3 CEN6 ARSH4 ampr (18) pAG419 pMSH2-R371S msh2-R371S HIS3 CEN6 ARSH4 ampr (18) pAG420 pMSH2-L521P msh2-L521P HIS3 CEN6 ARSH4 ampr (18) pAG86 pMSH2-D524Y msh2-D524Y HIS3 CEN6 ARSH4 ampr (18) pAG29 pMSH2-R542P msh2-R542P HIS3 CEN6 ARSH4 ampr (18) pAG421 pMSH2-A618V msh2-A618V HIS3 CEN6 ARSH4 ampr (18) pAG497 pMSH2-D621G msh2-D621G HIS3 CEN6 ARSH4 ampr This work pAG422 pMSH2-D621N msh2-D621N HIS3 CEN6 ARSH4 ampr (18) pAG31 pMSH2-P640L msh2-P640L HIS3 CEN6 ARSH4 ampr (18) pAG487 pMSH2-P640T msh2-P640T HIS3 CEN6 ARSH4 ampr This work pAG461 pMSH2-G711D msh2-G711D HIS3 CEN6 ARSH4 ampr (18) pAG239 pMSH2-G711R msh2-G711R HIS3 CEN6 ARSH4 ampr (18) pAG93 pMSH2-C716F msh2-C716F HIS3 CEN6 ARSH4 ampr (18) pAG211 pMSH2-C716R msh2-C716R HIS3 CEN6 ARSH4 ampr (18) pRS423 2µ HIS3 ampr (30) pAG122 pGAL-MSH2 PGAL10-MSH2 2µ HIS3 ampr (18) pAG508 pGAL-MSH2-L183P PGAL10-msh2-L183P 2µ HIS3 ampr This work pAG331 pGAL-MSH2-E194G PGAL10-msh2-E194G 2µ HIS3 ampr (18) pAG254 pGAL-MSH2-C195R PGAL10-msh2-C195R 2µ HIS3 ampr (18) pAG509 pGAL-MSH2-C195Y PGAL10-msh2-C195Y 2µ HIS3 ampr This work pAG561 pGAL-MSH2-C345F PGAL10-msh2-C345F 2µ HIS3 ampr This work pAG257 pGAL-MSH2-C345R PGAL10-msh2-C345R 2µ HIS3 ampr (18) pAG336 pGAL-MSH2-C345Y PGAL10-msh2-C345Y 2µ HIS3 ampr (18) pAG469 pGAL-MSH2-T347I PGAL10-msh2-T347I 2µ HIS3 ampr (18) pAG470 pGAL-MSH2-G350R PGAL10-msh2-G350R 2µ HIS3 ampr (18) pAG471 pGAL-MSH2-R371S PGAL10-msh2-R371S 2µ HIS3 ampr (18) pAG472 pGAL-MSH2-L521P PGAL10-msh2-L521P 2µ HIS3 ampr (18) pAG295 pGAL-MSH2-D524Y PGAL10-msh2-D524Y 2µ HIS3 ampr (18) pAG342 pGAL-MSH2-R542P PGAL10-msh2-R542P 2µ HIS3 ampr (18) pAG473 pGAL-MSH2-A618V PGAL10-msh2-A618V 2µ HIS3 ampr (18) pAG510 pGAL-MSH2-D621G PGAL10-msh2-D621G 2µ HIS3 ampr This work pAG474 pGAL-MSH2-D621N PGAL10-msh2-D621N 2µ HIS3 ampr (18) pAG349 pGAL-MSH2-P640L PGAL10-msh2-P640L 2µ HIS3 ampr (18) pAG513 pGAL-MSH2-P640T PGAL10-msh2-P640T 2µ HIS3 ampr This work pAG475 pGAL-MSH2-G711D PGAL10-msh2-G711D 2µ HIS3 ampr (18) pAG398 pGAL-MSH2-G711R PGAL10-msh2-G711R 2µ HIS3 ampr (18) pAG354 pGAL-MSH2-C716F PGAL10-msh2-C716F 2µ HIS3 ampr (18) pAG262 pGAL-MSH2-C716R PGAL10-msh2-C716R 2µ HIS3 ampr (18) pGBD-C2 GBD TRP1 2µ ampr (24) pAG124 pGBD-MSH2 GBD-MSH2 TRP1 2µ ampr (18)

7

Plasmid Number

Plasmid Name Relevant Markers Source

pAG491 pGBD-MSH2-L183P GBD-msh2-L183P TRP1 2µ ampr This work pAG378 pGBD-MSH2-E194G GBD-msh2-E194G TRP1 2µ ampr (18) pAG234 pGBD-MSH2-C195R GBD-msh2-C195R TRP1 2µ ampr (18) pAG504 pGBD-MSH2-C195Y GBD-msh2-C195Y TRP1 2µ ampr This work pAG518 pGBD-MSH2-C345F GBD-msh2-C345F TRP1 2µ ampr This work pAG235 pGBD-MSH2-C345R GBD-msh2-C345R TRP1 2µ ampr (18) pAG236 pGBD-MSH2-C345Y GBD-msh2-C345Y TRP1 2µ ampr (18) pAG432 pGBD-MSH2-T347I GBD-msh2-T347I TRP1 2µ ampr (18) pAG433 pGBD-MSH2-G350R GBD-msh2-G350R TRP1 2µ ampr (18) pAG446 pGBD-MSH2-R371S GBD-msh2-R371S TRP1 2µ ampr (18) pAG447 pGBD-MSH2-L521P GBD-msh2-L521P TRP1 2µ ampr (18) pAG130 pGBD-MSH2-D524Y GBD-msh2-D524Y TRP1 2µ ampr (18) pAG132 pGBD-MSH2-R542P GBD-msh2-R542P TRP1 2µ ampr (18) pAG448 pGBD-MSH2-A618V GBD-msh2-A618V TRP1 2µ ampr (18) pAG501 pGBD-MSH2-D621G GBD-msh2-D621G TRP1 2µ ampr This work pAG449 pGBD-MSH2-D621N GBD-msh2-D621N TRP1 2µ ampr (18) pAG135 pGBD-MSH2-P640L GBD-msh2-P640L TRP1 2µ ampr (18) pAG492 pGBD-MSH2-P640T GBD-msh2-P640T TRP1 2µ ampr This work pAG456 pGBD-MSH2-G711D GBD-msh2-G711D TRP1 2µ ampr (18) pAG265 pGBD-MSH2-G711R GBD-msh2-G711R TRP1 2µ ampr (18) pAG141 pGBD-MSH2-C716F GBD-msh2-C716F TRP1 2µ ampr (18) pAG215 pGBD-MSH2-C716R GBD-msh2-C716R TRP1 2µ ampr (18) pRG3077 pGBD-SAN1 GBD-SAN1 TRP1 2µ ampr (31) pRG1297 pGBD-san1C279S GBD-san1-C279S TRP1 2µ ampr (31) pAG568 pGAD-SAN1 GAD-SAN1 LEU2 2µ ampr This work pAG569 pGAD-san1C279S GAD-san1-C279S LEU2 2µ ampr This work pGAD-SAN1 and pGAD-san1C279S were created by ligating an EcoRI, BglII digested fragment of pRG3077 and pRG1297 respectively to the EcoRI, BglII digested pGAD-C2 backbone (24).

E194(E198)

C195(C199)

D621(D603)

A618(A600)

T347(T335)

G350(G336)

C345(C333)

Inner CoreL183(L187)

Central Core

Upper Core

R371(R359)

G711(G692)

P640(P622)

C716(C697)

Supplementary Figure 1: The amino acid positions important for maintaining steady-state levels of Msh2. The central image is of the hMutSα heterodimer structure. The relevant amino acid positions are indicated in black and enhanced with space-filling. The five structural domains are highlighted as follows: Domain 1 (blue), Domain 2 (green), Domain 3 (yellow), Domain 4, DNA binding (orange), DNA backbone (green thread), Domain 5, ATPase (red). The four stability clusters represented in Supplementary Table 1 are enlarged with arrows indicating the position within the heterodimer. Each amino acid is labeled with the yeast codon and the human codon below in parentheses in the enlarged images. Images were generated by manipulating 208E.pdb (32) using the Swiss PDB Viewer Version 4.0 (33).

D524(D506)

L521(L503)

R542(R524)

DNA binding

9

Supplementary Data: Cisplatin Sensitivity At 100, 200, and 400 µM of cisplatin, the average doubling times of the msh2-A618V strain

were 183±4, 205±5, and 252±9 minutes, respectively (n=13 trials). The average doubling times

of wild-type at these concentrations were 207±5, 238±5, and 282 ±8 minutes, respectively (n=15

trials). These doubling times were significantly different with p-values of < 0.001 (two-tailed t-

test). As additional examples, the average saturation point of msh2-A618V in 250 µM and 400

µM of cisplatin was 0.84±0.04 and 0.62±0.03 respectively (n=13 trials), in contrast to the

saturation points of wild type at 0.70±0.03 and 0.48±0.02 (n=15 trials). These saturation points,

with p-values of < 0.003 (two-tailed t-test) were significantly different.

10

Supplementary Methods

Mismatch Repair Viability Assay The haploid cells carry a mutation in the lagging strand DNA polymerase δ (encoded by POL3)

causing a defect in the proof-reading function. This allele of POL3 (pol3-01) causes cells to die

at elevated temperatures in combination with mutations in known mismatch repair genes.

(34-40). Without efficient mismatch repair cells expressing the error-prone polymerase are

overcome with mutations and cannot survive (35). In the strain background used in this study,

the combination of the pol3-01 allele and a deletion of MSH2 (msh2∆ pol3-01) is lethal at all

temperatures. To maintain viability, the strain was propagated with a wild type copy of MSH2

encoded on a URA3 centromere-based (CEN) plasmid [MSH2 URA3 CEN]. The plasmid shuffle

assays to test the mismatch repair in a pol3-01 background employed AGY1057, a msh2Δ pol3-

01 + pMSH2-MYC [MSH2-MYC:: KanMX6 URA3 CEN/ARS] strain transformed with HIS3

centromere-based plasmids pRS413 (vector), pMSH2 (wild-type MSH2), and the HIS3

centromere -based plasmids expressing the missense variants (e.g. pMSH2-D524Y). The strain

was also transformed with HIS3-based 2µ high-copy plasmids encoding PGAL10 driving

expression of MSH2 (pGAL-MSH2) or of the missense variants (e.g. pGAL-MSH2-D524Y) and

with the HIS3-based 2µ high-copy vector (pRS423). The cells with both plasmids were then

propagated selecting only for the HIS3 plasmids. Specifically, the cells were grown overnight in

medium lacking histidine with galactose (GAL) to activate PGAL10, with glucose (GLC) to repress

PGAL10, or with raffinose (RAF) that neither activates nor represses PGAL10. After propagation in

these conditions, the strains were plated onto medium with 5-fluoroorotic acid (5-FOA). 5-FOA

kills cells expressing URA3 and therefore selects against cells containing the URA3 plasmid with

wild-type MSH2. Under these conditions if the variant or wild-type version of MSH2 expressed

11

from the HIS3-based plasmid supported viability and the wild-type MSH2 URA3-based plasmid

was not needed for survival, then the cells displayed FOA resistance.

For the titration experiment, stains were pregrown overnight in medium with 2%

galactose to induce expression with varying percentages of glucose (0 to 2%) to titrate

expression. Selective pressure for the wild-type MSH2 URA3 plasmid was removed to allow for

plasmid loss during overnight growth. Serial dilutions of cells grown in synthetic medium

lacking histidine were spotted on plates containing synthetic medium lacking histidine, or

lacking histidine supplemented with 5-FOA. The plates also included either 2% galactose or

glucose as the carbon source. Plates were grown at 30oC for 2 days.

Turnover Assays Cells expressing the PGAL10 fusions were grown to logarithmic phase in synthetic medium with

2% raffinose. Cultures were diluted back to early logarithmic phase in the presence of synthetic

medium with 2% galactose and incubated for 3h to induce synthesis of MSH2 or the msh2

missense alleles. The cells were pelleted and resuspended in synthetic medium containing 2%

glucose to repress synthesis and maintained in logarithmic phase with dilutions of fresh medium.

At indicated time points (ranging from 0 to 6 h), 3 x 107 cells were pelleted, flash frozen in liquid

nitrogen, and stored at -80°C until processing for immunoblotting. The data were quantified and

corrected for growth rate as described in the supplementary material.

Growth Rate Correction in Turnover Experiments

During certain shut-off experiments cell division occurred complicating the turnover

analysis. Because transcription of MSH2 was repressed, each cell division diluted the Msh2 by

half. Therefore, the decrease in Msh2 signal is a combination of proteasomal degradation and

cell division. Additionally, cells treated with MG132, PMSF, and Bortezomib grew at a slower

12

rate than those cultures mock treated with DMSO. Thus correction for cell growth was required

for a more accurate comparison of the proteasome-mediated turnover rate. To correct for the

dilution due to cell division, we analyzed the turnover bands by densitometry, set the 0h point as

100% starting protein, and subsequent time points were divided by this initial value to produce a

percentage of Msh2. A trendline was imposed on this data to identify the exponential rate of

protein decay (due to both dilution and cellular degradation,

e−Rt ). We then plotted the OD600 of

the cultures versus time and identified the exponential rate at which the cells were growing (

eKt )

during the experiment. To isolate the turnover due purely to proteasomal degradation, these two

equations were multiplied to find the corrected values (

e(K−R )t ).

13

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