Supplementary Figure 4: In vitro GT mismatch repair assay | Nature Genetics

Supplementary Figure 4: In vitro GT mismatch repair assay

From: Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

Supplementary Figure 4

(a) A substrate containing a single GT mismatch was designed keeping a nick several hundred nucleotides away from the mismatch, as shown by the arrow. HindII restriction digestion, which cannot cleave the GT mismatch at its site but can efficiently cleave its repaired AT site, was used for measurement of mismatch repair ability. (b) Reactions were performed as described previously (Panigrahi et al., 2005). GT repaired products were digested by XmnI and HindIII. If repaired correctly, it would be sensitive to HindIII enzyme. The repaired products are marked by the brackets. The digested products were run on a 1% agarose gel and analyzed by Southern hybridization and quantification by Typhoon FLA 9500 Phosphorimager. The bar graphs represent three separate experiments. The last lane contains 50% of LoVo and 50% of MMR10 cell extracts. The protein concentration of the cell extracts was corrected for the purpose of quantification, and the graph was plotted accordingly.

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