Mutational. analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on t he similarity of MutH and PvuII structural models. The aims were to identif y DNA-binding residues; to determine whether MutH has the same mechanism fo r DNA binding and catalysis as PvuII; and to localize the residues responsi ble for MutH stimulation by MutL. No DNA-binding residues were identified i n the two flexible loop regions of MutH, although similar loops in PvuII ar e involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro, The results indicate that the MutH signal for DNA binding and cat alysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three pr esumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mut ations (MutH Delta 224 and MutH Delta 214) in the C-terminal end of the pro tein, localized the MutL stimulation region to five amino acids (Ala-220, L eu-221, Leu-222, Ala-223, and Arg-224).