The LexA repressor is inactivated when it catalyzes its own cleavage at a specific Ala–Gly peptide bond.

#Id: 2770

#Unit 13. Methods in Biology

DNA-binding proteins frequently perturb the DNA within the binding region, distorting the double helix. These perturbations are interesting, but are not generally detected by DNase footprinting because the protein keeps the DNase away.

#Id: 2771

#Unit 13. Methods in Biology

More detailed footprinting requires a smaller molecule that can

fit into the nooks and crannies of the DNA–protein complex and reveal more of the subtleties of the interaction. A favorite tool for this job is the methylating agent dimethyl sulfate (DMS).

#Id: 2772

#Unit 13. Methods in Biology

DMS footprinting follows a similar principle, except that we use the DNA methylating agent DMS, instead of DNase, to attack the DNA–protein complex. 

#Id: 2773

#Unit 13. Methods in Biology

In DMS footprinting, Unmethylated (or hypermethylated) sites show up on electrophoresis and demonstrate where the protein is bound to the DNA. 

#Id: 2774

#Unit 13. Methods in Biology

Methylation interference assay Dimethyl sulphate (DMS) methylates purines on DNA strand. Methylate DNA prior to adding protein.Protein binding is prevented at methylated sites

#Id: 2775

#Unit 13. Methods in Biology

Methylation interference assay Dimethyl sulphate (DMS) methylates purines on DNA strand. Methylate DNA prior to adding protein. Protein binding is prevented at methylated sites. After treatment and removal of protein of interest, treat with piperidine or NaOH which cuts strand at modified bases.