METHYL-DIRECTED MISMATCH REPAIR IS BIDIRECTIONAL

Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylate d d(GATC) sequence. This reaction is independent of the polarity of th e unmodified strand and can occur either 3' or 5' to the mismatch on t he unmethylated strand (Au, K. G., Welsh, K., and Modrich, P. (1992) J . Biol. Chem. 267, 12142-12148).The overall repair reaction also occur s without regard to polarity of the unmethylated strand. Both hemimeth ylated configurations of a linear heteroduplex containing a single d(G ATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system. Repair of both heterod uplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, an d DNA polymerase III holoenzyme, but the two substrates differ with re spect to exonuclease requirements for correction. When the unmethylate d d(GATC) sequence that directs repair is located 5' to the mismatch o n the unmodified strand, mismatch correction requires the 5' --> 3' hy drolytic activity of exonuclease VII or RecJ exonuclease. Repair direc ted by an unmodified d(GATC) sequence situated 3' to the mismatch depe nds on the 3' --> 5' activity of exonuclease I. Specific requirements for these activities are evident with circular heteroduplexes containi ng a single asymmetrically placed d(GATC) sequence, with the requireme nt for a 5' --> 3' or 3' --> 5' hydrolytic activity being determined b y the orientation of the unmethylated strand along the shorter path jo ining the two sites in the DNA circle. This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequen ce to direct correction. To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.