Construction of MMR Plasmid Substrates and Analysis of MMR Error Correction and Excision

Abstract

We describe simple and efficient construction of mismatch repair (MMR) substrates, by generation of gapped plasmids using one sequence-specific nicking endonuclease (N.BstNBI), ligation of synthetic oligomers into the gaps, and introduction of defined single nicks for initiation of MMR excision using a second such endonuclease (N.AlwI). We further describe measurement of completed mismatch correction and a sensitive quantitative assay for MMR excision intermediates. These methods can be easily adapted for construction of substrates containing defined DNA lesions, for analysis of MMR responses to DNA damage and for studies of other DNA repair pathways.