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• W2070742295 abstract "Previous biochemical analysis ofEscherichia 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. Previous biochemical analysis ofEscherichia 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. methyl-directed mismatch repair single-strand DNA rifampicin kilobase pair(s) The correction of DNA polymerase misincorporation errors plays an important role in the maintenance of genetic integrity. DNA biosynthetic errors in Escherichia coli are corrected by the methyl-directed mismatch repair (MMR)1 system, which removes incorrect nucleotides by a strand-specific excision reaction that is directed to daughter DNA strands by virtue of the transient absence of d(GATC) methylation within newly synthesized DNA (1Meselson M. Low K.B. The Recombination of Genetic Material. Academic Press, Inc., San Diego, CA1988: 91-113Crossref Google Scholar). This system processes both base-base and small insertion/deletion mismatches (2Su S.S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar, 3Parker B.O. Marinus M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1730-1734Crossref PubMed Scopus (181) Google Scholar, 4Fang W. Wu J.Y. Su M.J. J. Biol. Chem. 1997; 272: 22714-22720Crossref PubMed Scopus (25) Google Scholar). Inactivation of the genes encoding the nonessential MutH, MutL, MutS, or UvrD components confers a large increase (100–500-fold) in mutation rate (1Meselson M. Low K.B. The Recombination of Genetic Material. Academic Press, Inc., San Diego, CA1988: 91-113Crossref Google Scholar, 5Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar).Biochemical analysis of the repair of artificial mismatch-containing substrates has defined 10 activities in a methyl-directed excision repair reaction reconstituted with purified components (6Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar, 7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). Repair is initiated by the binding of MutS to the mismatch (2Su S.S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar, 3Parker B.O. Marinus M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1730-1734Crossref PubMed Scopus (181) Google Scholar, 8Su S.S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar), with MutL binding to the heteroduplex in a MutS- and ATP-dependent manner (9Grilley M. Welsh K.M. Su S.S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar, 10Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 11Galio L. Bouquet C. Brooks P. Nucleic Acids Res. 1999; 27: 2325-2331Crossref PubMed Scopus (62) Google Scholar). Assembly of this ternary complex is sufficient to activate the d(GATC) endonuclease activity of MutH, which incises the unmethylated strand of a hemimethylated d(GATC) sequence (12Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar), as well as the unwinding activity of DNA helicase II (uvrD/mutU gene product), which enters the helix at the incised d(GATC) sequence and unwinds toward the mismatch (13Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9202-9207Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). That portion of the unwound, incised strand is subject to degradation by one of several single-strand DNA (ssDNA) exonucleases (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar, 14Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar), and DNA removed in this manner is resynthesized by DNA polymerase III holoenzyme in the presence of single-strand DNA-binding protein. Finally, DNA ligase restores covalent continuity to the repaired strand (6Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar).In vitro excision can be directed by an incised d(GATC) site that is located either 5′ or 3′ to the mismatch on the unmethylated strand, and the nature of the exonuclease requirement depends on the relative orientation of the two DNA sites (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). If the initial incision is made 5′ to the mismatch, excision requires either ExoVII or RecJ exonuclease, both of which are capable of 5′→3′ hydrolysis of ssDNA (15Chase J.W. Richardson C.C. J. Biol. Chem. 1974; 249: 4553-4561Abstract Full Text PDF PubMed Google Scholar, 16Lovett S.T. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2627-2631Crossref PubMed Scopus (219) Google Scholar), whereas exonuclease I, which is capable of 3′→5′ hydrolytic activity (17Lehman I.R. Nussbaum A.L. J. Biol. Chem. 1964; 239: 2628-2636Abstract Full Text PDF PubMed Google Scholar), is sufficient to meet the exonuclease requirement when d(GATC) incision occurs 3′ to the mispair. Extracts prepared from a RecJ− ExoVII− double mutant are defective in repair directed by a 5′ d(GATC) sequence. However, extracts of ExoI-deficient strains retain repair activity on heteroduplexes where repair is directed by a d(GATC) site located 3′ to the mismatch, suggesting that at least one additional activity can meet the 3′ exonuclease requirement (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar).The phenotypes of strains mutant in RecJ, ExoI, and/or ExoVII exonucleases have implicated these genes and their products in DNA repair and recombination (18Kushner S.R. Nagaishi H. Templin A. Clark A.J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 824-847Crossref PubMed Scopus (227) Google Scholar, 19Chase J.W. Richardson C.C. J. Bacteriol. 1977; 129: 934-947Crossref PubMed Google Scholar, 20Lovett S.T. Clark A.J. J. Bacteriol. 1984; 157: 190-196Crossref PubMed Google Scholar). In addition, ExoI and ExoVII also appear to have overlapping roles in preventing frameshift and quasi-palindrome templated mutations, two classes of mutations associated with strand slippage during DNA replication (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 22Viswanathan M. Lacirignola J.J. Hurley R.L. Lovett S.T. J. Mol. Biol. 2000; 302: 553-564Crossref PubMed Scopus (56) Google Scholar). Despite the biochemical evidence implicating ExoI, RecJ, and ExoVII in mismatch repair (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), triple mutants deficient in the three activities fail to show enhanced mutability in assays that score for spontaneous base substitutions, a hallmark of MMR deficiency (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 23Harris R.S. Ross K.J. Lombardo M.J. Rosenberg S.M. J. Bacteriol. 1998; 180: 989-993Crossref PubMed Google Scholar). However, it seems unlikely that DNA helicase II alone would be sufficient to support methyl-directed excision, especially when one considers that excision tracts can be a thousand nucleotides or more (14Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar, 24Wagner Jr., R. Meselson M. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4135-4139Crossref PubMed Scopus (193) Google Scholar). Inasmuch as ExoI, RecJ, and ExoVII have been implicated in a UV-induced recombination pathway that also depends on MutH, MutL, and MutS mismatch repair activities (25Feng W.Y. Hays J.B. Genetics. 1995; 140: 1175-1186Crossref PubMed Google Scholar), and because biochemical experiments have suggested involvement of at least one additional exonuclease in the methyl-directed reaction, we have sought the identity of other exonucleases that might support methyl-directed excision.A novel E. coli 3′→5′ exonuclease, exonuclease X, has recently been identified (26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This activity hydrolyzes both single- and double-strand DNA, but its affinity for single-strand DNA is approximately 10 times greater than that for duplex DNA. Unlike RecJ, ExoI, and ExoVII, which are processive exonucleases, ExoX is distributive, hydrolyzing only one or a few nucleotides before releasing from its substrate. The fact that ExoX is distributive does not affect its potency as a nuclease; in fact, its affinity for single-strand DNA and its specific activity rival those of RecJ and ExoI.We demonstrate here that ExoX also supports methyl-directed mismatch repair in vitro. The results show that either RecJ or ExoVII is sufficient to meet the exonuclease requirement when excision is initiated 5′ to the mispair, whereas either ExoI, ExoVII, or ExoX is sufficient to meet the exonuclease requirement when excision is initiated 3′ to the mismatch. Simultaneous inactivation of ExoX, ExoI, RecJ, and ExoVII abolishes normal mismatch repair in vitrobut confers only a modest increase in mutation rate. We provide evidence elsewhere (27Burdett V. Baitinger C. Viswanathan M. Lovett S.T. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6765-6770Crossref PubMed Scopus (175) Google Scholar) that these exonucleases participate in methyl-directed repair in vivo, and we demonstrate that the relatively low mutability of the quadruple exonuclease mutant is due to under-recovery of mutants as a consequence of lethal events triggered by the occurrence of mismatches in this genetic background.DISCUSSIONPrevious work has implicated E. coli ExoX, as well as the ssDNA exonucleases ExoI, ExoVII, and RecJ, in the repair of UV-damaged DNA (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have now demonstrated that these ssDNA exonucleases also function redundantly during mismatch correction. Inactivation of ExoI, ExoVII, ExoX, and RecJ conferred a moderate mutator phenotype, and the corresponding cell-free extracts were devoid of normal mismatch repair activity in vitro on model heteroduplexes.The modest increase in mutability observed in the absence of these four activities is less than that observed for other blocks of the MMR pathway, including mutS, mutL, mutH, or uvrDmutant strains. There are two possible explanations for this paradox. The simple heteroduplexes used for biochemical assay may not be good models for natural substrates. Consequently, the four exonucleases studied here may process some but not all mismatches in the cell. The alternate possibility is that all four exonucleases contribute to most, if not all, methyl-directed repair events—the relatively low mutability of the exonuclease-deficient strain may be due to under-recovery of mutants due to lethal events triggered by the occurrence of mismatches in this genetic background. We present evidence elsewhere (27Burdett V. Baitinger C. Viswanathan M. Lovett S.T. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6765-6770Crossref PubMed Scopus (175) Google Scholar) that the latter explanation is likely to be correct. Quadruple mutants deficient in the four exonucleases are cold-sensitive for growth, undergo filamentation, and are extremely sensitive to 2-aminopurine, a base analogue that promotes mispairing and triggers the methyl-directed system (31Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (270) Google Scholar). These effects are suppressed by the introduction of null mutations in mutS, mutL, mutH, or uvrD, genes that mediate the earlier steps in the MMR pathway. The exact nature of this lethality is not known, but presumably accumulation of ssDNA displaced during MMR is deleterious to the cell.Depending on the relative position of the mispair to the incision site, different exonucleases are recruited for the excision step of MMR. ExoI, ExoX, and ExoVII are all capable of participating in the 3′ repair pathway. Both ExoI and ExoX are strict 3′→5′ exonucleases (26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,32Chase J.W. Richardson C.C. J. Biol. Chem. 1974; 249: 4545-4552Abstract Full Text PDF PubMed Google Scholar), and repair of the 3′ substrate in extracts deficient in these two exonucleases is reduced by 50%. No repair activity above background was detected in extracts when a null mutation in ExoVII was introduced into the ExoI− ExoX− deficient background. The results of this study differ from our earlier finding with respect to the ability of ExoVII to support repair of a 3′ heteroduplex. Whereas previous work failed to detect 3′ heteroduplex repair supported by ExoVII in the reconstituted system (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we now find that ExoVII can support 3′ heteroduplex repair in both cell-free extracts and the purified system. However, in agreement with the previous study, we find that ExoVII is much less effective in supporting 3′ heteroduplex repair as compared with its activity with 5′ substrates (Fig. 4, comparelanes 3 and 7). In agreement with earlier results (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we show here that RecJ or ExoVII is sufficient for removal of mismatched bases via excision tracts initiated 5′ of the mismatch. Thus both the 3′ and 5′ exonuclease activities of ExoVII can be utilized in the mismatch correction system.These findings imply redundancy of exonuclease involvement in this mutation avoidance pathway and the level of repair supported by any one of these exonuclease activities is apparently sufficient to maintain a low level of spontaneous mutability. Mismatch repair proficiency is exhibited in the absence of all but one of these four exonucleases, further demonstrating that mismatch excision in vivo is truly bidirectional and can be accomplished from a single direction if necessary.Although recJ and xseA (ExoVII large subunit) orthologues can be found in almost all bacterial genomes sequenced to date, the other exonucleases are apparently more restricted in their distribution. This means that, at least in bacteria, a different assortment of exonucleases is likely to be called into play for the excision step of MMR in different species. The redundancy of exonucleases in E. coli may be due to the specialized roles these enzymes play in other aspects of DNA metabolism. For instance, RecJ mediates recombination via the RecF pathway (20Lovett S.T. Clark A.J. J. Bacteriol. 1984; 157: 190-196Crossref PubMed Google Scholar) and may process stalled replication forks (33Courcelle J. Hanawalt P.C. Mol. Gen. Genet. 1999; 262: 543-551Crossref PubMed Scopus (215) Google Scholar). RecJ and ExoI also assist recombination via the RecBCD pathway (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 34Miesel L. Roth J.R. J. Bacteriol. 1996; 178: 3146-3155Crossref PubMed Google Scholar, 35Razavy H. Szigety S.K. Rosenberg S.M. Genetics. 1996; 142: 333-339Crossref PubMed Google Scholar, 36Friedman-Ohana R. Cohen A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6909-6914Crossref PubMed Scopus (26) Google Scholar). ExoI and ExoVII play important roles in the avoidance of misalignment errors during replication, such as frameshifts (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar) and quasi-palindrome templated mutations (22Viswanathan M. Lacirignola J.J. Hurley R.L. Lovett S.T. J. Mol. Biol. 2000; 302: 553-564Crossref PubMed Scopus (56) Google Scholar). The correction of DNA polymerase misincorporation errors plays an important role in the maintenance of genetic integrity. DNA biosynthetic errors in Escherichia coli are corrected by the methyl-directed mismatch repair (MMR)1 system, which removes incorrect nucleotides by a strand-specific excision reaction that is directed to daughter DNA strands by virtue of the transient absence of d(GATC) methylation within newly synthesized DNA (1Meselson M. Low K.B. The Recombination of Genetic Material. Academic Press, Inc., San Diego, CA1988: 91-113Crossref Google Scholar). This system processes both base-base and small insertion/deletion mismatches (2Su S.S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar, 3Parker B.O. Marinus M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1730-1734Crossref PubMed Scopus (181) Google Scholar, 4Fang W. Wu J.Y. Su M.J. J. Biol. Chem. 1997; 272: 22714-22720Crossref PubMed Scopus (25) Google Scholar). Inactivation of the genes encoding the nonessential MutH, MutL, MutS, or UvrD components confers a large increase (100–500-fold) in mutation rate (1Meselson M. Low K.B. The Recombination of Genetic Material. Academic Press, Inc., San Diego, CA1988: 91-113Crossref Google Scholar, 5Modrich P. Annu. Rev. Genet. 1991; 25: 229-253Crossref PubMed Scopus (771) Google Scholar). Biochemical analysis of the repair of artificial mismatch-containing substrates has defined 10 activities in a methyl-directed excision repair reaction reconstituted with purified components (6Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar, 7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). Repair is initiated by the binding of MutS to the mismatch (2Su S.S. Lahue R.S. Au K.G. Modrich P. J. Biol. Chem. 1988; 263: 6829-6835Abstract Full Text PDF PubMed Google Scholar, 3Parker B.O. Marinus M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1730-1734Crossref PubMed Scopus (181) Google Scholar, 8Su S.S. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5057-5061Crossref PubMed Scopus (258) Google Scholar), with MutL binding to the heteroduplex in a MutS- and ATP-dependent manner (9Grilley M. Welsh K.M. Su S.S. Modrich P. J. Biol. Chem. 1989; 264: 1000-1004Abstract Full Text PDF PubMed Google Scholar, 10Allen D.J. Makhov A. Grilley M. Taylor J. Thresher R. Modrich P. Griffith J.D. EMBO J. 1997; 16: 4467-4476Crossref PubMed Scopus (268) Google Scholar, 11Galio L. Bouquet C. Brooks P. Nucleic Acids Res. 1999; 27: 2325-2331Crossref PubMed Scopus (62) Google Scholar). Assembly of this ternary complex is sufficient to activate the d(GATC) endonuclease activity of MutH, which incises the unmethylated strand of a hemimethylated d(GATC) sequence (12Au K.G. Welsh K. Modrich P. J. Biol. Chem. 1992; 267: 12142-12148Abstract Full Text PDF PubMed Google Scholar), as well as the unwinding activity of DNA helicase II (uvrD/mutU gene product), which enters the helix at the incised d(GATC) sequence and unwinds toward the mismatch (13Dao V. Modrich P. J. Biol. Chem. 1998; 273: 9202-9207Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). That portion of the unwound, incised strand is subject to degradation by one of several single-strand DNA (ssDNA) exonucleases (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar, 14Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar), and DNA removed in this manner is resynthesized by DNA polymerase III holoenzyme in the presence of single-strand DNA-binding protein. Finally, DNA ligase restores covalent continuity to the repaired strand (6Lahue R.S. Au K.G. Modrich P. Science. 1989; 245: 160-164Crossref PubMed Scopus (444) Google Scholar). In vitro excision can be directed by an incised d(GATC) site that is located either 5′ or 3′ to the mismatch on the unmethylated strand, and the nature of the exonuclease requirement depends on the relative orientation of the two DNA sites (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). If the initial incision is made 5′ to the mismatch, excision requires either ExoVII or RecJ exonuclease, both of which are capable of 5′→3′ hydrolysis of ssDNA (15Chase J.W. Richardson C.C. J. Biol. Chem. 1974; 249: 4553-4561Abstract Full Text PDF PubMed Google Scholar, 16Lovett S.T. Kolodner R.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2627-2631Crossref PubMed Scopus (219) Google Scholar), whereas exonuclease I, which is capable of 3′→5′ hydrolytic activity (17Lehman I.R. Nussbaum A.L. J. Biol. Chem. 1964; 239: 2628-2636Abstract Full Text PDF PubMed Google Scholar), is sufficient to meet the exonuclease requirement when d(GATC) incision occurs 3′ to the mispair. Extracts prepared from a RecJ− ExoVII− double mutant are defective in repair directed by a 5′ d(GATC) sequence. However, extracts of ExoI-deficient strains retain repair activity on heteroduplexes where repair is directed by a d(GATC) site located 3′ to the mismatch, suggesting that at least one additional activity can meet the 3′ exonuclease requirement (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar). The phenotypes of strains mutant in RecJ, ExoI, and/or ExoVII exonucleases have implicated these genes and their products in DNA repair and recombination (18Kushner S.R. Nagaishi H. Templin A. Clark A.J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 824-847Crossref PubMed Scopus (227) Google Scholar, 19Chase J.W. Richardson C.C. J. Bacteriol. 1977; 129: 934-947Crossref PubMed Google Scholar, 20Lovett S.T. Clark A.J. J. Bacteriol. 1984; 157: 190-196Crossref PubMed Google Scholar). In addition, ExoI and ExoVII also appear to have overlapping roles in preventing frameshift and quasi-palindrome templated mutations, two classes of mutations associated with strand slippage during DNA replication (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 22Viswanathan M. Lacirignola J.J. Hurley R.L. Lovett S.T. J. Mol. Biol. 2000; 302: 553-564Crossref PubMed Scopus (56) Google Scholar). Despite the biochemical evidence implicating ExoI, RecJ, and ExoVII in mismatch repair (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), triple mutants deficient in the three activities fail to show enhanced mutability in assays that score for spontaneous base substitutions, a hallmark of MMR deficiency (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 23Harris R.S. Ross K.J. Lombardo M.J. Rosenberg S.M. J. Bacteriol. 1998; 180: 989-993Crossref PubMed Google Scholar). However, it seems unlikely that DNA helicase II alone would be sufficient to support methyl-directed excision, especially when one considers that excision tracts can be a thousand nucleotides or more (14Grilley M. Griffith J. Modrich P. J. Biol. Chem. 1993; 268: 11830-11837Abstract Full Text PDF PubMed Google Scholar, 24Wagner Jr., R. Meselson M. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4135-4139Crossref PubMed Scopus (193) Google Scholar). Inasmuch as ExoI, RecJ, and ExoVII have been implicated in a UV-induced recombination pathway that also depends on MutH, MutL, and MutS mismatch repair activities (25Feng W.Y. Hays J.B. Genetics. 1995; 140: 1175-1186Crossref PubMed Google Scholar), and because biochemical experiments have suggested involvement of at least one additional exonuclease in the methyl-directed reaction, we have sought the identity of other exonucleases that might support methyl-directed excision. A novel E. coli 3′→5′ exonuclease, exonuclease X, has recently been identified (26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This activity hydrolyzes both single- and double-strand DNA, but its affinity for single-strand DNA is approximately 10 times greater than that for duplex DNA. Unlike RecJ, ExoI, and ExoVII, which are processive exonucleases, ExoX is distributive, hydrolyzing only one or a few nucleotides before releasing from its substrate. The fact that ExoX is distributive does not affect its potency as a nuclease; in fact, its affinity for single-strand DNA and its specific activity rival those of RecJ and ExoI. We demonstrate here that ExoX also supports methyl-directed mismatch repair in vitro. The results show that either RecJ or ExoVII is sufficient to meet the exonuclease requirement when excision is initiated 5′ to the mispair, whereas either ExoI, ExoVII, or ExoX is sufficient to meet the exonuclease requirement when excision is initiated 3′ to the mismatch. Simultaneous inactivation of ExoX, ExoI, RecJ, and ExoVII abolishes normal mismatch repair in vitrobut confers only a modest increase in mutation rate. We provide evidence elsewhere (27Burdett V. Baitinger C. Viswanathan M. Lovett S.T. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6765-6770Crossref PubMed Scopus (175) Google Scholar) that these exonucleases participate in methyl-directed repair in vivo, and we demonstrate that the relatively low mutability of the quadruple exonuclease mutant is due to under-recovery of mutants as a consequence of lethal events triggered by the occurrence of mismatches in this genetic background. DISCUSSIONPrevious work has implicated E. coli ExoX, as well as the ssDNA exonucleases ExoI, ExoVII, and RecJ, in the repair of UV-damaged DNA (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have now demonstrated that these ssDNA exonucleases also function redundantly during mismatch correction. Inactivation of ExoI, ExoVII, ExoX, and RecJ conferred a moderate mutator phenotype, and the corresponding cell-free extracts were devoid of normal mismatch repair activity in vitro on model heteroduplexes.The modest increase in mutability observed in the absence of these four activities is less than that observed for other blocks of the MMR pathway, including mutS, mutL, mutH, or uvrDmutant strains. There are two possible explanations for this paradox. The simple heteroduplexes used for biochemical assay may not be good models for natural substrates. Consequently, the four exonucleases studied here may process some but not all mismatches in the cell. The alternate possibility is that all four exonucleases contribute to most, if not all, methyl-directed repair events—the relatively low mutability of the exonuclease-deficient strain may be due to under-recovery of mutants due to lethal events triggered by the occurrence of mismatches in this genetic background. We present evidence elsewhere (27Burdett V. Baitinger C. Viswanathan M. Lovett S.T. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6765-6770Crossref PubMed Scopus (175) Google Scholar) that the latter explanation is likely to be correct. Quadruple mutants deficient in the four exonucleases are cold-sensitive for growth, undergo filamentation, and are extremely sensitive to 2-aminopurine, a base analogue that promotes mispairing and triggers the methyl-directed system (31Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (270) Google Scholar). These effects are suppressed by the introduction of null mutations in mutS, mutL, mutH, or uvrD, genes that mediate the earlier steps in the MMR pathway. The exact nature of this lethality is not known, but presumably accumulation of ssDNA displaced during MMR is deleterious to the cell.Depending on the relative position of the mispair to the incision site, different exonucleases are recruited for the excision step of MMR. ExoI, ExoX, and ExoVII are all capable of participating in the 3′ repair pathway. Both ExoI and ExoX are strict 3′→5′ exonucleases (26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,32Chase J.W. Richardson C.C. J. Biol. Chem. 1974; 249: 4545-4552Abstract Full Text PDF PubMed Google Scholar), and repair of the 3′ substrate in extracts deficient in these two exonucleases is reduced by 50%. No repair activity above background was detected in extracts when a null mutation in ExoVII was introduced into the ExoI− ExoX− deficient background. The results of this study differ from our earlier finding with respect to the ability of ExoVII to support repair of a 3′ heteroduplex. Whereas previous work failed to detect 3′ heteroduplex repair supported by ExoVII in the reconstituted system (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we now find that ExoVII can support 3′ heteroduplex repair in both cell-free extracts and the purified system. However, in agreement with the previous study, we find that ExoVII is much less effective in supporting 3′ heteroduplex repair as compared with its activity with 5′ substrates (Fig. 4, comparelanes 3 and 7). In agreement with earlier results (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we show here that RecJ or ExoVII is sufficient for removal of mismatched bases via excision tracts initiated 5′ of the mismatch. Thus both the 3′ and 5′ exonuclease activities of ExoVII can be utilized in the mismatch correction system.These findings imply redundancy of exonuclease involvement in this mutation avoidance pathway and the level of repair supported by any one of these exonuclease activities is apparently sufficient to maintain a low level of spontaneous mutability. Mismatch repair proficiency is exhibited in the absence of all but one of these four exonucleases, further demonstrating that mismatch excision in vivo is truly bidirectional and can be accomplished from a single direction if necessary.Although recJ and xseA (ExoVII large subunit) orthologues can be found in almost all bacterial genomes sequenced to date, the other exonucleases are apparently more restricted in their distribution. This means that, at least in bacteria, a different assortment of exonucleases is likely to be called into play for the excision step of MMR in different species. The redundancy of exonucleases in E. coli may be due to the specialized roles these enzymes play in other aspects of DNA metabolism. For instance, RecJ mediates recombination via the RecF pathway (20Lovett S.T. Clark A.J. J. Bacteriol. 1984; 157: 190-196Crossref PubMed Google Scholar) and may process stalled replication forks (33Courcelle J. Hanawalt P.C. Mol. Gen. Genet. 1999; 262: 543-551Crossref PubMed Scopus (215) Google Scholar). RecJ and ExoI also assist recombination via the RecBCD pathway (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 34Miesel L. Roth J.R. J. Bacteriol. 1996; 178: 3146-3155Crossref PubMed Google Scholar, 35Razavy H. Szigety S.K. Rosenberg S.M. Genetics. 1996; 142: 333-339Crossref PubMed Google Scholar, 36Friedman-Ohana R. Cohen A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6909-6914Crossref PubMed Scopus (26) Google Scholar). ExoI and ExoVII play important roles in the avoidance of misalignment errors during replication, such as frameshifts (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar) and quasi-palindrome templated mutations (22Viswanathan M. Lacirignola J.J. Hurley R.L. Lovett S.T. J. Mol. Biol. 2000; 302: 553-564Crossref PubMed Scopus (56) Google Scholar). Previous work has implicated E. coli ExoX, as well as the ssDNA exonucleases ExoI, ExoVII, and RecJ, in the repair of UV-damaged DNA (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We have now demonstrated that these ssDNA exonucleases also function redundantly during mismatch correction. Inactivation of ExoI, ExoVII, ExoX, and RecJ conferred a moderate mutator phenotype, and the corresponding cell-free extracts were devoid of normal mismatch repair activity in vitro on model heteroduplexes. The modest increase in mutability observed in the absence of these four activities is less than that observed for other blocks of the MMR pathway, including mutS, mutL, mutH, or uvrDmutant strains. There are two possible explanations for this paradox. The simple heteroduplexes used for biochemical assay may not be good models for natural substrates. Consequently, the four exonucleases studied here may process some but not all mismatches in the cell. The alternate possibility is that all four exonucleases contribute to most, if not all, methyl-directed repair events—the relatively low mutability of the exonuclease-deficient strain may be due to under-recovery of mutants due to lethal events triggered by the occurrence of mismatches in this genetic background. We present evidence elsewhere (27Burdett V. Baitinger C. Viswanathan M. Lovett S.T. Modrich P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6765-6770Crossref PubMed Scopus (175) Google Scholar) that the latter explanation is likely to be correct. Quadruple mutants deficient in the four exonucleases are cold-sensitive for growth, undergo filamentation, and are extremely sensitive to 2-aminopurine, a base analogue that promotes mispairing and triggers the methyl-directed system (31Glickman B.W. Radman M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1063-1067Crossref PubMed Scopus (270) Google Scholar). These effects are suppressed by the introduction of null mutations in mutS, mutL, mutH, or uvrD, genes that mediate the earlier steps in the MMR pathway. The exact nature of this lethality is not known, but presumably accumulation of ssDNA displaced during MMR is deleterious to the cell. Depending on the relative position of the mispair to the incision site, different exonucleases are recruited for the excision step of MMR. ExoI, ExoX, and ExoVII are all capable of participating in the 3′ repair pathway. Both ExoI and ExoX are strict 3′→5′ exonucleases (26Viswanathan M. Lovett S.T. J. Biol. Chem. 1999; 274: 30094-30100Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,32Chase J.W. Richardson C.C. J. Biol. Chem. 1974; 249: 4545-4552Abstract Full Text PDF PubMed Google Scholar), and repair of the 3′ substrate in extracts deficient in these two exonucleases is reduced by 50%. No repair activity above background was detected in extracts when a null mutation in ExoVII was introduced into the ExoI− ExoX− deficient background. The results of this study differ from our earlier finding with respect to the ability of ExoVII to support repair of a 3′ heteroduplex. Whereas previous work failed to detect 3′ heteroduplex repair supported by ExoVII in the reconstituted system (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we now find that ExoVII can support 3′ heteroduplex repair in both cell-free extracts and the purified system. However, in agreement with the previous study, we find that ExoVII is much less effective in supporting 3′ heteroduplex repair as compared with its activity with 5′ substrates (Fig. 4, comparelanes 3 and 7). In agreement with earlier results (7Cooper D.L. Lahue R.S. Modrich P. J. Biol. Chem. 1993; 268: 11823-11829Abstract Full Text PDF PubMed Google Scholar), we show here that RecJ or ExoVII is sufficient for removal of mismatched bases via excision tracts initiated 5′ of the mismatch. Thus both the 3′ and 5′ exonuclease activities of ExoVII can be utilized in the mismatch correction system. These findings imply redundancy of exonuclease involvement in this mutation avoidance pathway and the level of repair supported by any one of these exonuclease activities is apparently sufficient to maintain a low level of spontaneous mutability. Mismatch repair proficiency is exhibited in the absence of all but one of these four exonucleases, further demonstrating that mismatch excision in vivo is truly bidirectional and can be accomplished from a single direction if necessary. Although recJ and xseA (ExoVII large subunit) orthologues can be found in almost all bacterial genomes sequenced to date, the other exonucleases are apparently more restricted in their distribution. This means that, at least in bacteria, a different assortment of exonucleases is likely to be called into play for the excision step of MMR in different species. The redundancy of exonucleases in E. coli may be due to the specialized roles these enzymes play in other aspects of DNA metabolism. For instance, RecJ mediates recombination via the RecF pathway (20Lovett S.T. Clark A.J. J. Bacteriol. 1984; 157: 190-196Crossref PubMed Google Scholar) and may process stalled replication forks (33Courcelle J. Hanawalt P.C. Mol. Gen. Genet. 1999; 262: 543-551Crossref PubMed Scopus (215) Google Scholar). RecJ and ExoI also assist recombination via the RecBCD pathway (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar, 34Miesel L. Roth J.R. J. Bacteriol. 1996; 178: 3146-3155Crossref PubMed Google Scholar, 35Razavy H. Szigety S.K. Rosenberg S.M. Genetics. 1996; 142: 333-339Crossref PubMed Google Scholar, 36Friedman-Ohana R. Cohen A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6909-6914Crossref PubMed Scopus (26) Google Scholar). ExoI and ExoVII play important roles in the avoidance of misalignment errors during replication, such as frameshifts (21Viswanathan M. Lovett S.T. Genetics. 1998; 149: 7-16Crossref PubMed Google Scholar) and quasi-palindrome templated mutations (22Viswanathan M. Lacirignola J.J. Hurley R.L. Lovett S.T. J. Mol. Biol. 2000; 302: 553-564Crossref PubMed Scopus (56) Google Scholar). We thank Dr. Mike O'Donnell for the generous gift of DNA polymerase III holoenzyme and W. Wackernagel, R. Kolodner, and M. Marinus for strains." @default.
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