Abstract
A method for measuring repair in extracts of human cells of DNA containing mismatched bases is described. Wild-type and mutant M13mp2 phage derivatives are used to prepare a double-stranded DNA substrate that contains a nick in the (−) strand and mismatched bases in the lacZα-complementation gene. Introduction of this heteroduplex DNA into an Escherichia coli strain defective in mismatch repair yields M13 plaques on host indicator plates that are blue, colorless, or a mixture of the two. Repair of the same substrate during incubation in a human cell extract reduces the percentage of mixed-color plaques and increases the percentage of pure color plaques. Since the nick directs repair to the (−) strand, the ratio of the (+) strand phenotype increases relative to that of the (−) strand phenotype. The assay permits determination of the efficiency of repair of all single-base mismatches in a variety of sequence contexts as well as repair of substrates containing one or more unpaired bases in either strand. It can be used to examine repair in extracts made from a variety of cell lines. Substrates can also be analyzed for repair-specific DNA synthesis by including radioactive dNTP precursors in the extract reaction mixture.
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