Purification of Tth-Muts

Purification of T. thermophilus MutS protein

Frozen cells (100 g) were thawed, suspended in buffer I [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM β-mercaptoethanol and 25% (w/v) sucrose] and disrupted by sonication on ice. Brij-58 was added to a final concentration of 0.5% (w/v) and then the cell extract was stirred for 30 min at 4°C. Following this, the cell lysate was incubated at 70° C for 10 min and centrifuged (15 000 g) for 40 min at 4°C. A DEAE-cellulose DE52 column (bed volume 500 ml) was equilibrated with buffer II [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM β-mercaptoethanol and 10% (v/v) glycerol] before the supernatant was loaded. The column was then washed once with 150 ml of buffer II and eluted with a 3000 ml gradient of 0–500 mM NaCl in buffer II. Ammonium sulfate was added to the MutS fraction to give a final concentration of 15% (w/v) and then it was loaded onto a Phenyl-Toyopearl 650M column (bed volume 60 ml) previously equilibrated with 15% (w/v) ammonium sulfate in buffer II. The column was washed with 50 ml of the same buffer and then eluted with a 600 ml gradient of 15–0% (w/v) ammonium sulfate in buffer II. Fractions containing the protein were loaded onto a Sephacryl S-300 HR column (bed volume 400 ml) previously equilibrated with buffer III [50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM β-mercaptoethanol and 10% (v/v) glycerol]. The column was eluted with buffer III, the fractions containing MutS protein were concentrated and then stored at 4°C. At each step, the column fractionation pattern was analyzed by SDS-PAGE. The final amount of purified MutS protein was 40 mg and the yield was 0.4 mg protein/g cells.

The N-terminal amino acid sequence of the purified protein was analyzed using an automated protein sequencer (ABI, model 473A). The molar extinction coefficient of T.thermophilus MutS protein was calculated to be 51 000 M−1cm−1 at an absorption maximum of ∼278 nm

Mismatch DNA binding assay

Mismatch DNA binding was measured by gel retardation assay. Complementary strands of a 16mer or a 37mer oligonucleotide were synthesized (Fig. 6). Each top strand was labeled at the 5′ end with [γ-32P]ATP using with polynucleotide kinase. The oligonucleotides were annealed at 70°C (16mer) or 95°C (37mer) for 10 min, allowed to cool to 30°C (at 1°C/min), and then placed on ice until required. When the assay was carried out using 37 bp duplex DNA as a substrate, each binding assay reaction mixture (total volume 10µl), comprising 0.2 pmol oligonucleotide duplex, the assay buffer [50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM β-mercaptoethanol, 10% (v/v) glycerol and 20 mM MgCl2] and T.thermophilus MutS protein, was incubated at 35 or 60°C for 30 min. After adding 2 µl 50% (w/v) sucrose, the binding assay reaction mixtures were loaded onto a 6% (w/v) acrylamide gel containing 20 mM magnesium acetate and run at 35°C for 90 min or 60°C for 70 min at 76 V in 1× TAE buffer (15) containing 20 mM magnesium acetate. The gel was then dried and placed in contact with an imaging plate. The bands were analyzed using a BAS2000 image analyzer. When the assay was carried out using 16 bp duplex DNA, concentration of magnesium ion was adjusted to 5 mM in the assay buffer and 1 mM in the running buffer, respectively.

ATPase assay

TEA  buffer:                    2L/1L of 1X:       50 mL of 10X:
50 mM triethanolamine-HCl (TEA) 18.57 g/9.29 g     4.64 g
50 mM KCl                       7.46 g/3.73 g      1.86 g
20 mM MgCl2*6H2O                8.13 g/ 4.07 g     2.03 g
pH 7.5

ATP:,100 mM, in H2O

5 mL: 0.275 g

pH 7.5 (pH changes quickly!)

Ammonium molybdate: 4.2%, in 4 N HCl 50 mL

2.1 g (NH4)6Mo7O24

16.51 mL of 12.1 N HCl

H3PO4, 1000 µM, in 1 N H2SO4

50 mL: 58 µL of 85% H3PO4

1.38 mL of 36 N H2SO4

Add H2O to 50 mL

Citric acid 34%, in H2O

50 mL: 17 g

H2SO4, 0.01 N, in H2O

50 mL: 138 µL of 36 N

Triton X-100, 1%, in H2O

50 mL: 0.5 mL of 100%

Malachite green 0.045%, in H2O

36 mL: 16.2 mg

Make dye solution (make day of experiment, make fresh every time):

• Combine 36 mL of 0.045% malachite green, 12 mL of 4.2% ammonium molybdate, and 1 mL of 1% Triton X-100
• Let dye solution sit on bench top at least 1 hour
• Filter through 0.45 µm filter (with dye recipe as written, can do one PO4 standard curve and seven protein combinations)
• After filtering, let dye solution sit on bench top 1 hour before using

Phosphate Std

Final PO4 conc (uM)	uL of 1000 µM H3PO4 (in 1 N H2SO4)	uL of 0.01 N H2SO4
0	0	1000
20	20	980
40	40	960
60	60	940
80	80	920
100	100	900
120	120	880
140	140	860
160	160	840
180	180	820
200	200	800

Perform phosphate standard curve:

a. Blank spec at 660 nm with 1 mL H2O

b. Combine 800 µL dye solution and 50 µL of 1X TEA in eleven 1.5 mL tubes

c. Add 50 µL of phosphate standard solution, start timer, vortex, incubate 60 sec

d. At 1 minute, add 100 µL of 34% citric acid, vortex, pipette into cuvette

e. Take three readings at 660 nm at 1 min 25 sec, 1 min 30 sec, 1 min 35 sec

f. Pour liquid from cuvette into waste container

g. Repeat steps c-f for all standards

Perform steady-state ATPase assay with proteins:

a. Thaw ATP aliquot, keep at room temp

b. Combine:

i. 255 µL H2O

ii. 37.5 µL of 10X TEA buffer

iii. 37.5 µL of 1.25 µM protein (final 0.125 µM)

iv. 37.5 µL of 3 µM protein (final 0.3 µM) or 37.5 µL of 1X TEA buffer

c. Vortex, let incubate at room temp for 10 minutes

d. During incubation, prepare 7 identical tubes with 800 µL dye solution and 50 µL of 1X TEA

e. After 10 minute incubation is completed, add 7.5 µL of 100 mM ATP to reaction, vortex, start timer (final 2 mM)

f. At 1 min 55 sec (5 seconds before timepoint), remove 50 µL of reaction, at 2 min add it to dye solution tube, vortex, let react for 1 min

g. At 3 minutes, add 100 µL of 34% citric acid to tube, vortex, pipette into cuvette

h. Take 3 readings at 660 nm at 3 min 25 sec, 3 min 30 sec, 3 min 35 sec

i. Repeat steps f, g, and h at 4, 6, 8, 10, 12, and 14 minutes