Abstract
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.
Highlights
The correction of DNA polymerase misincorporation errors plays an important role in the maintenance of genetic integrity
E. coli Deficient in ExoI, ExoVII, ExoX, and RecJ Exonucleases Display a Modest Mutator Phenotype—Previous work implicated three single-strand DNA (ssDNA)-specific exonucleases (ExoI, RecJ, and ExoVII) in methyl-directed mismatch repair in vitro and indicated that at least one additional activity participates in the pathway [7]
To examine the possible role of exonuclease X in this regard, an isogenic set of mutant strains was constructed with null alleles of ExoX and the three ssDNA-specific exonucleases that are known to participate in mismatch correction (Table I)
Summary
Bacterial Strains, Media, and Antibiotics—Isogenic strains listed in Table I, which were derived from BT199, were constructed by P1 phagemediated transduction [28]. Mutation Assays—Mutation assay scoring for Rif resistance was performed as described previously [21]. In Vitro Mismatch Repair Assays—E. coli extracts were prepared as described previously [7] Briefly, ammonium sulfate pellets were resuspended in 25 mM HEPES (potassium salt, pH 7.6), 0.1 mM EDTA, 2 mM dithiothreitol, and 50 mM KCl and dialyzed against this buffer until the conductivity of the sample was equivalent to that of 25 mM HEPES buffer containing 150 mM KCl. Mismatch repair in cell-free extracts was performed at 37 °C in 20 mM Tris-HCl buffered reactions (pH 7.6) containing 90 mM KCl (final concentration including the contribution from the extract) as described previously [7], except that reactions (20 l) contained 24 fmol of heteroduplex and ϳ0.1 mg of extract protein. Reactions were performed in a 20 mM Tris-HCl buffer (pH 8) with a reduced salt concentration (55 mM KCl). Reactions contained (per 20 l) 24 fmol of heteroduplex DNA, 35 ng of MutS, 30 ng of MutL, 0.26 ng of MutH, 3 ng of DNA helicase II, 150 ng of single-stranded DNA-binding protein (SSB), 20 ng of E. coli DNA ligase, 100 ng of DNA polymerase III holoenzyme (generously provided by Dr Mike O’Donnell, Rockefeller University), and exonucleases as indicated
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