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, R. S., and Modrich, P. (1993) J. Biol. Chem. 268, 11823-11829). We show here that ExoX also participates in methyl-directed mismatch repair. Analysis of the reaction with crude extracts and purified components demonstrated that ExoX can mediate repair directed from a strand signal 3' of a mismatch. Whereas extracts of all possible single, double, and triple exonuclease mutants displayed significant residual mismatch repair, extracts deficient in RecJ, ExoI, ExoVII, and ExoX exonucleases were devoid of normal repair activity. The RecJ(-) ExoVII(-) ExoI(-) ExoX(-) strain displayed a 7-fold increase in mutation rate, a significant increase, but less than that observed for other blocks of the mismatch repair pathway. This elevation is epistatic to deficiency for MutS, suggesting an effect via the mismatch repair pathway. Our other work (Burdett, V., Baitinger, C., Viswanathan, M., Lovett, S. T., and Modrich, P. (2001) Proc. 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 that is triggered by mismatched base pairs in this genetic background. The availability of any one exonuclease is enough to support full mismatch correction, as evident from the normal mutation rates of all triple mutants. Because three of these exonucleases possess a strict polarity of digestion, this suggests that mismatch repair can occur exclusively from a 3' or a 5' direction to the mismatch, if necessary.
Viswanathan
M
M
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Burdett
V
V
Baitinger
C
C
Modrich
P
P
Lovett
S T
ST
D001426:Bacterial Proteins / Q000502:physiology*; D020137:Base Pair Mismatch; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000502:physiology*; D009154:Mutation
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases; C489467:RecJ protein, E coli; C070765:recJ protein, Bacteria; C020133:exodeoxyribonuclease III; C026870:exodeoxyribonuclease VII