Redundant exonuclease involvement in Escherichia coli methyl-directed mismatch repair

Previous biochemical analysis of Escherichia coli methyl-directed mismatch repair implicates three redundant single-strand DNA-specific exonucleases ( RecJ, ExoI, and ExoVII) and at least one additional unknown exonuclease in the excision reaction (Cooper, D. L., Lahue, IL S., and Modrich, P. (1993) J. BioL Chem. 268, 11823-11829). We show here that ExoX also participates i n methyl-directed mismatch repair. Analysis of the reaction with crude extr acts and purified components demonstrated that ExoX can mediate repair dire cted from a strand signal 3 ' of a mismatch. Whereas extracts of all possib le single, double, and triple exonuclease mutants displayed significant res idual mismatch repair, extracts deficient in RecJ, ExoI, ExoVII, and ExoX e xonucleases were devoid of normal repair activity. The RecJ(-) ExoVII(-) Ex oI(-) ExoX(-) strain displayed a 7-fold increase in mutation rate, a signif icant increase, but less than that observed for other blocks of the mismatc h repair pathway. This elevation is epistatic to deficiency for MutS, sugge sting an effect via the mismatch repair pathway. Our other work (Burdett, V ., Baitinger, C., Viswanathan, M., Lovett, S. T., and Modrich, P. (2001) Pr oc. Natl. Acad. Sci. U. S. A 98, 6765-6770) suggests that mutants are under -recovered in the exonuclease-deficient strain due to loss of viability tha t is triggered by mismatched base pairs in this genetic background. The ava ilability of any one exonuclease is enough to support full mismatch correct ion, as evident from the normal mutation rates of all triple mutants. Becau se three of these exonucleases possess a strict polarity of digestion, this suggests that mismatch repair can occur exclusively from a 3 ' or a 5 ' di rection to the mismatch, if necessary.