SUPPLEMENTARY INFORMATION. The nucleotide binding dynamics of MSH2/MSH3 are lesion-dependent.

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1 SUPPLEMENTARY INFORMATION The nucleotide binding dynamics of /MSH are lesion-dependent. Barbara A. L. Owen, Walter H. Lang, and Cynthia T. McMurray*

2 mau mau mau mau ml ml ml ml a A 28 A 28 A 28 A AMP-PNP AMP ADP ATP AMP-PNP Retention Time (minutes) ANIS, Bodipy-AMP-PNP b Owen Supplemental Fig. 1 Phosphokinase, PEP /MSH6, µm Supplemental Fig. 1. Characterization or treatments of unlabeled adenine nucleotides. (a) Mono-Q (Akta) chromatography of unlabeled adenine nucleotides is shown as plots of A 28 (ma) versus retention time (minutes). Nucleotide, or analogue, is as indicated on each chromatogram. The gradient in each chromatogram was from 25 to 6 mm NH 4 HCO, and 1. µmol of each nucleotide was applied to a 5 ml Mono-Q column. (b) Plot of data with anisotropy versus /MSHh6 concentration with either untreated AMP-PNP(+Mg 2+ ) (closed circles,-) or AMP- PNP(+Mg 2+ ) treated with an ATP regeneration system (open circles, +) consisting of Pyruvate kinase and PEP (phosphoenol pyruvate). Experimental details are found in supplemental methods.

3 a MSH6 b MSH Owen Supplemental Fig. 2 Supplemental Fig. 2. Densitometry of ADP-MSH cross-linked subunits. (a) Scans of SDS-PAGE (reduced) gels visualizing [α 2 P]ADP(+Mg 2+ ) cross-linked to human /MSH6 subunits, as indicated. Area under peaks was determined using Image J software (NIH) and results plotted versus ADP concentration to determine relative binding affinities (K d ). (b) Same as for (a), except [α 2 P]ADP(+Mg 2+ ) was cross-linked to human /MSH and individual MSH subunits are as indicated.

4 Owen Supplemental Fig. D 2 D or 2 D D M BD-ADP Dissociation 4 δanisotropy (ADP) (AMP-PNP) Time, min [AMP-PNP], [ADP]= 5 µm Supplemental Fig.. Kinetics of BD-ADP dissociation from h/msh after competition with unlabeled ADP or unlabeled AMP-PNP by fluorescence anisotropy. (top) Schematic of experimental design for ADP dissociation. Unlabeled ADP (gray ball, D) or unlabeled AMP-PNP (white ball, M) at 5µM is added as a competitor to BD-ADP-bound-/MSH. Dissociation of BD-ADP (Red ball, D) is followed as the loss of anisotropy with time. (bottom) Results of (top) plotted as the change in anisotropy versus time for BD-ADP bound to /MSH after competition with 5 µm unlabeled ADP (black filled circles) or 5 µm unlabeled AMP-PNP (while open circles). Scans and quantification were determined as described previously. The resulting K d values for ATP (-Mg 2+ ) with DNA are listed in Supplemental Table 1a.

5 /MSH6 Owen Supplemental Fig. 4 [ATP], µm No DNA MSH6 [ATP], µm +G/C G C [ATP], µm +G/T G T MSH6 MSH6 Supplemental Fig 4. DNA has minimal effect on ATP binding affinity for /MSH6. SDS-PAGE gels of human /MSH6 (1 nm) cross-linked to [α 2 P]ATP(-Mg 2+ ) at concentrations as indicated either in the absence of added DNA (top panel), or with 1. µm homoduplex (+G/C) DNA (middle panel) or 1. µm single base mismatched (+G/T) DNA (lower panel). or MSH6 bands are shown and densitometric scans and quantification were determined as described previously. The resulting K d values for ATP (-Mg 2+ ) with DNA are listed in Supplemental Table 1a.

6 Owen Supplemental Fig. 5 K d µm [α 2 P]ADP(+Mg 2+ ), µm MSH Template No DNA MSH G/C G C (CA) Supplemental Fig. 5. DNA binding influences the nucleotide binding affinity of ADP for the subunits of /MSH. Representative gels resolving the [α 2 P]ADP(+Mg 2+ ) x-linked products in the absence of DNA (No DNA), pre-bound to homoduplex DNA (+G/C), or pre-bound to the (CA) 4 heteroduplex loop (+(CA) 4 ). The concentration of nucleotides (µm) is indicated. The K d (µm) are indicated.

7 a 2 T DT 2 or 2 T 2 T b c K d µm MSH >12 /MSH + [α 2 P]ATP(-Mg 2+ ), µm +G/C /MSH + [α 2 P]AMP-PNP(+Mg 2+ ), µm +G/C Template +(CA) 4 Template +(CA) 4 No DNA G C No DNA G C MSH MSH Supplemental Fig. 6. DNA binding influences the nucleotide binding affinity of ATP and AMP-PNP for the subunits of /MSH. Same as supplemental Fig. 5 except for ATP (b) or AMP-PNP (c) is the added nucleotide. Owen Supplemental Fig. 6

8 Nucleotide, pmol, Hexokinase ADP Owen Supplemental Fig. 7 ATP Supplemental Fig. 7. Conversion of [ 2 P]ATP(+Mg 2+ ) to [ 2 P]ADP(+Mg 2+ ) by treatment with Hexokinase. Thin layer chromatography of [ 2 P]ATP(+Mg 2+ ) before (lanes 1-) and after (lanes 4-6) treatment with Hexokinase at concentrations (nmolar) listed at top. Reaction and chromatography conditions are specified in supplemental methods.

9 Table 1. Nucleotide and DNA binding affinities of MSH complexes Table 1a. Nucleotide binding affinities (x-linking), K d (µm) Subunit Ligand no DNA +G/C +G/T ADP(+Mg 2+ ).65 ±.8 ATP (-Mg).55 ±.8.2 ±.6.7 ±.4 MSH6 ADP(+Mg 2+ ) NQ* ATP (-Mg).8 ±.14.2 ±.7.2 ±.6 Subunit Ligand K d (µm) no DNA +G/C +(CA) 4 -loop ADP(+Mg 2+ ).21 ±.4.19 ±.6.2 ±.11 ATP (-Mg).2 ± ± ±.49 (+Mg 2+ ) AMP-PNP 1.7 ± ± ± 2.6 MSH ADP(+Mg 2+ ).29 ± ±.4.4 ±.5 ATP (-Mg).29 ± ± ± 6.6 (+Mg 2+ ) AMP-PNP 1.72 ± ±. >12 NQ*, not quantifiable Table 1b. Nucleotide binding affinities (anisotropy), K d (µm) -MSH6 BD-ADP.2 ±.2 BD-ATP (-Mg).9 ±.14 BD-AMP-PNP.2 ±. -MSH BD-ADP.26 ±.4 BD-ATP (-Mg).62 ±.21 BD-AMP-PNP.47 ±.2 Table 1c. ADP-MSH competitor K i (µm) -MSH6 ADP.29 ±.4 ATP (-Mg) 2.2 ±.5 -MSH ADP.7 ±.2 ATP (-Mg) 1.96 ±.2 Table 1d. DNA binding affinities (anisotropy), K d (nm) -MSH6-(G/T) No nucleotide 8.8 ±.15 -MSH-(CA 4 -loop) No nucleotide 1.8 ± 1.4 ADP 6.8 ± 1.4 -MSH-(G/C)* none > 19 *(G/C), determined by competition, see supplemental methods.

10 SUPPLEMENTAL METHODS Pyruvate Kinase treatment of bodipy-amp-pnp Bodipy-AMP-PNP was treated with an ATPase regeneration system to convert any possible ADP contamination to ATP and anisotropy measured. Pyruvate kinase, at 75 µg/ml, was incubated with Bodipy-AMP-PNP in a buffer containing 1 mm phosphoenol pyruvate, 2 mm HEPES, ph 6.8, 15 mm K-Acetate, 5 mm Mg-Acetate, and 25 mm sorbitol, overnight on ice. Mono-Q analysis of unlabeled nucleotides All nucleotide preparations were reconstituted from lyophilized reagents in de-ionized water to concentrations of 1-1 mm immediately before use and then discarded. Characterization of each batch of nucleotides was performed by Mono-Q chromatography using a mono-q (5/5) column (Amersham/GE Healthcare). One micromole of each nucleotide was applied and eluted from the column with a gradient from 1 mm to 6 mm NH 4 HC. Elution profiles are shown in Supplemental Figure 2A. Protein Purification, and His-tagged MSH or His-tagged MSH6 were over-expressed in SF9 insect cells using a pfastbac dual expression system (GIBCO-BRL). The original, MSH, and MSH6 clones were a gift from Josef Jiricny. SF9 insect cell pellets expressing the recombinant proteins were obtained under contract with the University Colorado Cancer Center s Cell Culture Core Facility (Denver, CO). All purification steps were performed at 4 C. Cells expressing recombinant /MSH or /MSH6 complex were resuspended in Lysis buffer (25 mm HepesNaOH, ph 8.1, mm NaCl, 2 mm Imidazole, 1% glycerol (v/v) containing a protease inhibitor cocktail (Roche))

11 and lysed by repeated passage through a 25 gauge needle as described (Wilson, 1999). After centrifugation for 1 hr 2 min at 4,g, the supernatant was loaded onto a 5 ml HiTrap chelating column (GE Healthcare) charged with nickel nitrilotriacetic acid affinity column and equilibrated with Lysis buffer. The bound proteins were then eluted with a 25 ml 2 to 2 mm imidazole gradient. The peak fractions containing the /MSH or /MSH6 complex eluted at 14 mm imidazole and were then loaded onto a MonoP and HiTrap Heparin column (both GE Healthcare) connected in tandem and equilibrated in column buffer (25mM HepesNaOH, ph 8.1,.1 mm EDTA, 1% glycerol (v/v), 1 mm DTT) containing mm NaCl. After eluting the unbound proteins, the MonoP column was disconnected from the Heparin column and /MSH6 complex was eluted from MonoP with a 2 ml mm to 1M NaCl gradient. The protein complex eluted at 57 mm NaCl. Proteins bound to the Heparin column were eluted with a NaCl gradient from mm to 1 M. The /MSH proteins eluted at 45 mm NaCl. The /MSH containing fractions were then applied to MonoQ (GE Healthcare) equilibrated in column buffer containing 1 mm NaCl and eluted with a 2 ml gradient form 1 mm to 1 M NaCl. The /MSH complex eluted at 2 mm NaCl. Both /MSH and /MSH6 containing fractions were then adjusted to 2% glycerol (v/v), aliquoted and frozen at -8 C. Purified proteins were assessed for purity and degradation by SDS-PAGE and Western blot analysis and judged to be greater than 99% pure and intact. The heparin-sepharose column removed the bound nucleotide, and the absence of pre-bound nucleotides, either for ADP indirectly or ATP directly, was assessed using a sensitive luciferase assay for ATP as reported previously (Lamers, 2). Both ADP and ATP levels from the purified proteins were less than 1%.