Preparation of nuclear extracts
The nuclear extracts were prepared according to Dignam et al..
Electrophoretic Mobility Shift Assay (EMSA)
All EMSA reactions were carried out in binding buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 1 mM DTT, 1 mg/ml BSA, 5% glycerol) in a volume of 5 µl using the unspecific competitor poly(dI-dC) (Sigma-Aldrich). 20 ng of poly(dI-dC) was used for 100 ng of recombinant MutS⍺ (purified as described in) and 1 µg for 10 µg of nuclear extracts. For all EMSAs, 10 fmol of [α-32P]-dNTP labeled, or [γ-32P]-ATP phosphorylated oligos were used. For the supershift assays, 200 ng of antibody (Anti-MSH6; BD Biosciences 610919) and 1 mM ATP were added, together with the proteins where indicated. Proteins, and antibodies where indicated, were incubated for 20 min on ice in binding buffer. Labeled DNA was added and the mixture was left at RT for another 20 min before loading on a 5% polyacrylamide gel (Acryl/ Bis 29:1, Amresco) eluted with 1x TAE (40 mM Tris, 20 mM acetate, 1 mM EDTA). The gels were run at 200 V for 1 h in 1x TAE and dried in a gel dryer for 1 h. The gels were exposed to phosphor screens and the autoradiographs were developed in a Typhoon FLA 9500 (GE Healthcare Life Sciences).
Oligo 1 upper 5' TATTCCTGGTCAGCGTGACCGGAGC 3'
and lower 5'TTCAGCTCCGGTCACGCTGACCAGGAATA 3',
oligo 2 upper 5'GATTTTCTTTATTCGCCGTGAAGAGAATTTATG3'
and lower 5'CATAAATTCTCTTCACGGCGAATAAAGAAA3',
oligo 3 upper 5'GCAGAAAACAGCCACGTGTTCCTGAAC3'
and lower 5' GTTCAGGAACACGTGGCTGTTTTC3',
T/G oligo upper 5' CCAGACGTCTGTTGACGTTGGGAAGCTTGAG 3'
and lower 5'CTCAAGCTTCCCAACGTCGACAGACGTCTGG 3'. The bold, underlined Ts in oligo 1 were replaced by BrdU to enable crosslinking.
SDS-PAGE of cross-linked binding reactions
EMSAs were performed as described and cross-linked for 5 min (~720 mJ) in a UV Stratalinker 1800 (Stratagene). Samples were taken up in 2x SDS loading buffer and boiled for 5 min before loading on a 7.5 % denaturing polyacrylamide gel and run for 1–2 h in 10 % SDS-running buffer at 130 V. Gels were dried for 1 h, exposed to phosphor screens and developed in a Typhoon FLA 9500 (GE Healthcare).
In vitro mismatch repair assay
The substrate was a circular heteroduplex of 3196 bp, which contained either a C, a mC, or a T opposite a G within the SalI site at position 42, as well as a single Nt.BstNBI nick in the upper (C, mC, or T) strand at position 308. It was incubated with nuclear extracts of HEK293 cells supplemented with dGTP, dCTP, dTTP and [a-32P]dATP in a buffer containing 20 mM Tris HCl pH 7.6, 110 mM KCl, 5 mM MgCl2, 1 mM glutathione, 1.5 mM ATP, 50 μg/ml BSA. Reactions were incubated at 30°C for 25 min, then incubated in a stop solution (0.5 mmol/l EDTA, 1.5% SDS, 2.5 mg/ml proteinase K) for 1 h. The DNA was purified using Qiagen MinElute Reaction Cleanup Kit, followed by digestion with DraI and SalI. The repair synthesis step can be visualized by the incorporation of the radiolabeled nucleotide. In the case of 100% efficient repair, all radioactivity should be found predominantly in the 1516 bp band, with some also in the 1307 bp band.
The restriction fragments were visualized under UV and incorporation of the radioactive nucleotide was detected on the dried gel using the Typhoon FLA 9500 PhosphorImager.