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. 1998 Mar 2;17(5):1535–1541. doi: 10.1093/emboj/17.5.1535

Evidence for a physical interaction between the Escherichia coli methyl-directed mismatch repair proteins MutL and UvrD.

M C Hall 1, J R Jordan 1, S W Matson 1
PMCID: PMC1170501  PMID: 9482750

Abstract

UvrD (DNA helicase II) is an essential component of two major DNA repair pathways in Escherichia coli: methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. In addition, it has an undefined role in the RecF recombination pathway and possibly in replication. In an effort to better understand the role of UvrD in these various aspects of DNA metabolism, a yeast two-hybrid screen was used to search for interacting protein partners. Screening of an E.coli genomic library revealed a potential interaction between UvrD and MutL, a component of the methyl-directed mismatch repair pathway. The interaction was confirmed by affinity chromatography using purified proteins. Deletion analysis demonstrated that the C-terminal 218 amino acids (residues 398-615) of MutL were sufficient to produce the two-hybrid interaction with UvrD. On the other hand, both the N- and C-termini of UvrD were required for interaction with MutL. The implications of this interaction for the mismatch repair mechanism are discussed.

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Selected References

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  1. Allen D. J., Makhov A., Grilley M., Taylor J., Thresher R., Modrich P., Griffith J. D. MutS mediates heteroduplex loop formation by a translocation mechanism. EMBO J. 1997 Jul 16;16(14):4467–4476. doi: 10.1093/emboj/16.14.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arthur H. M., Lloyd R. G. Hyper-recombination in uvrD mutants of Escherichia coli K-12. Mol Gen Genet. 1980;180(1):185–191. doi: 10.1007/BF00267368. [DOI] [PubMed] [Google Scholar]
  3. Au K. G., Welsh K., Modrich P. Initiation of methyl-directed mismatch repair. J Biol Chem. 1992 Jun 15;267(17):12142–12148. [PubMed] [Google Scholar]
  4. Bende S. M., Grafström R. H. The DNA binding properties of the MutL protein isolated from Escherichia coli. Nucleic Acids Res. 1991 Apr 11;19(7):1549–1555. doi: 10.1093/nar/19.7.1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brosh R. M., Jr, Matson S. W. A point mutation in Escherichia coli DNA helicase II renders the enzyme nonfunctional in two DNA repair pathways. Evidence for initiation of unwinding from a nick in vivo. J Biol Chem. 1997 Jan 3;272(1):572–579. doi: 10.1074/jbc.272.1.572. [DOI] [PubMed] [Google Scholar]
  6. Brosh R. M., Jr, Matson S. W. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol. 1995 Oct;177(19):5612–5621. doi: 10.1128/jb.177.19.5612-5621.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Caron P. R., Kushner S. R., Grossman L. Involvement of helicase II (uvrD gene product) and DNA polymerase I in excision mediated by the uvrABC protein complex. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4925–4929. doi: 10.1073/pnas.82.15.4925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper D. L., Lahue R. S., Modrich P. Methyl-directed mismatch repair is bidirectional. J Biol Chem. 1993 Jun 5;268(16):11823–11829. [PubMed] [Google Scholar]
  9. Eshleman J. R., Markowitz S. D. Microsatellite instability in inherited and sporadic neoplasms. Curr Opin Oncol. 1995 Jan;7(1):83–89. [PubMed] [Google Scholar]
  10. Feinstein S. I., Low K. B. Hyper-recombining recipient strains in bacterial conjugation. Genetics. 1986 May;113(1):13–33. doi: 10.1093/genetics/113.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fishel R., Kolodner R. D. Identification of mismatch repair genes and their role in the development of cancer. Curr Opin Genet Dev. 1995 Jun;5(3):382–395. doi: 10.1016/0959-437x(95)80055-7. [DOI] [PubMed] [Google Scholar]
  12. George J. W., Brosh R. M., Jr, Matson S. W. A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II. A biochemical and genetic characterization. J Mol Biol. 1994 Jan 14;235(2):424–435. doi: 10.1006/jmbi.1994.1003. [DOI] [PubMed] [Google Scholar]
  13. Georgi-Geisberger P., Hoffmann-Berling H. Direction of the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase II. Eur J Biochem. 1990 Sep 24;192(3):689–693. doi: 10.1111/j.1432-1033.1990.tb19277.x. [DOI] [PubMed] [Google Scholar]
  14. Glickman B. W., Radman M. Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1063–1067. doi: 10.1073/pnas.77.2.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grilley M., Griffith J., Modrich P. Bidirectional excision in methyl-directed mismatch repair. J Biol Chem. 1993 Jun 5;268(16):11830–11837. [PubMed] [Google Scholar]
  16. Grilley M., Welsh K. M., Su S. S., Modrich P. Isolation and characterization of the Escherichia coli mutL gene product. J Biol Chem. 1989 Jan 15;264(2):1000–1004. [PubMed] [Google Scholar]
  17. Hall M. C., Matson S. W. Mutation of a highly conserved arginine in motif IV of Escherichia coli DNA helicase II results in an ATP-binding defect. J Biol Chem. 1997 Jul 25;272(30):18614–18620. doi: 10.1074/jbc.272.30.18614. [DOI] [PubMed] [Google Scholar]
  18. Hoeijmakers J. H. Nucleotide excision repair I: from E. coli to yeast. Trends Genet. 1993 May;9(5):173–177. doi: 10.1016/0168-9525(93)90164-d. [DOI] [PubMed] [Google Scholar]
  19. Horii Z., Clark A. J. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol. 1973 Oct 25;80(2):327–344. doi: 10.1016/0022-2836(73)90176-9. [DOI] [PubMed] [Google Scholar]
  20. Husain I., Van Houten B., Thomas D. C., Abdel-Monem M., Sancar A. Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6774–6778. doi: 10.1073/pnas.82.20.6774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jiricny J. Mismatch repair and cancer. Cancer Surv. 1996;28:47–68. [PubMed] [Google Scholar]
  22. Karran P., Bignami M. DNA damage tolerance, mismatch repair and genome instability. Bioessays. 1994 Nov;16(11):833–839. doi: 10.1002/bies.950161110. [DOI] [PubMed] [Google Scholar]
  23. Kolodner R. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev. 1996 Jun 15;10(12):1433–1442. doi: 10.1101/gad.10.12.1433. [DOI] [PubMed] [Google Scholar]
  24. Kramer W., Kramer B., Williamson M. S., Fogel S. Cloning and nucleotide sequence of DNA mismatch repair gene PMS1 from Saccharomyces cerevisiae: homology of PMS1 to procaryotic MutL and HexB. J Bacteriol. 1989 Oct;171(10):5339–5346. doi: 10.1128/jb.171.10.5339-5346.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunkel T. A. DNA-mismatch repair. The intricacies of eukaryotic spell-checking. Curr Biol. 1995 Oct 1;5(10):1091–1094. doi: 10.1016/s0960-9822(95)00218-1. [DOI] [PubMed] [Google Scholar]
  26. Lahue R. S., Au K. G., Modrich P. DNA mismatch correction in a defined system. Science. 1989 Jul 14;245(4914):160–164. doi: 10.1126/science.2665076. [DOI] [PubMed] [Google Scholar]
  27. Lechner R. L., Richardson C. C. A preformed, topologically stable replication fork. Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. J Biol Chem. 1983 Sep 25;258(18):11185–11196. [PubMed] [Google Scholar]
  28. Lu A. L., Clark S., Modrich P. Methyl-directed repair of DNA base-pair mismatches in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4639–4643. doi: 10.1073/pnas.80.15.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matson S. W. DNA helicases of Escherichia coli. Prog Nucleic Acid Res Mol Biol. 1991;40:289–326. doi: 10.1016/s0079-6603(08)60845-4. [DOI] [PubMed] [Google Scholar]
  30. Matson S. W. Escherichia coli helicase II (urvD gene product) translocates unidirectionally in a 3' to 5' direction. J Biol Chem. 1986 Aug 5;261(22):10169–10175. [PubMed] [Google Scholar]
  31. Matson S. W., Richardson C. C. DNA-dependent nucleoside 5'-triphosphatase activity of the gene 4 protein of bacteriophage T7. J Biol Chem. 1983 Nov 25;258(22):14009–14016. [PubMed] [Google Scholar]
  32. Mellon I., Champe G. N. Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1292–1297. doi: 10.1073/pnas.93.3.1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mendonca V. M., Kaiser-Rogers K., Matson S. W. Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli. J Bacteriol. 1993 Aug;175(15):4641–4651. doi: 10.1128/jb.175.15.4641-4651.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mendonca V. M., Klepin H. D., Matson S. W. DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair. J Bacteriol. 1995 Mar;177(5):1326–1335. doi: 10.1128/jb.177.5.1326-1335.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Modrich P., Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem. 1996;65:101–133. doi: 10.1146/annurev.bi.65.070196.000533. [DOI] [PubMed] [Google Scholar]
  36. Morel P., Hejna J. A., Ehrlich S. D., Cassuto E. Antipairing and strand transferase activities of E. coli helicase II (UvrD). Nucleic Acids Res. 1993 Jul 11;21(14):3205–3209. doi: 10.1093/nar/21.14.3205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nevers P., Spatz H. C. Escherichia coli mutants uvr D and uvr E deficient in gene conversion of lambda-heteroduplexes. Mol Gen Genet. 1975 Aug 27;139(3):233–243. doi: 10.1007/BF00268974. [DOI] [PubMed] [Google Scholar]
  38. Prolla T. A., Christie D. M., Liskay R. M. Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene. Mol Cell Biol. 1994 Jan;14(1):407–415. doi: 10.1128/mcb.14.1.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pukkila P. J., Peterson J., Herman G., Modrich P., Meselson M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics. 1983 Aug;104(4):571–582. doi: 10.1093/genetics/104.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Runyon G. T., Bear D. G., Lohman T. M. Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6383–6387. doi: 10.1073/pnas.87.16.6383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Runyon G. T., Wong I., Lohman T. M. Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II). Biochemistry. 1993 Jan 19;32(2):602–612. doi: 10.1021/bi00053a028. [DOI] [PubMed] [Google Scholar]
  42. Sancar A. DNA excision repair. Annu Rev Biochem. 1996;65:43–81. doi: 10.1146/annurev.bi.65.070196.000355. [DOI] [PubMed] [Google Scholar]
  43. Schellhorn H. E., Low K. B. Indirect stimulation of recombination in Escherichia coli K-12: dependence on recJ, uvrA, and uvrD. J Bacteriol. 1991 Oct;173(19):6192–6198. doi: 10.1128/jb.173.19.6192-6198.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Su S. S., Lahue R. S., Au K. G., Modrich P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J Biol Chem. 1988 May 15;263(14):6829–6835. [PubMed] [Google Scholar]
  45. Su S. S., Modrich P. Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5057–5061. doi: 10.1073/pnas.83.14.5057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Taucher-Scholz G., Hoffmann-Berling H. Identification of the gene for DNA helicase II of Escherichia coli. Eur J Biochem. 1983 Dec 15;137(3):573–580. doi: 10.1111/j.1432-1033.1983.tb07864.x. [DOI] [PubMed] [Google Scholar]
  47. Umar A., Kunkel T. A. DNA-replication fidelity, mismatch repair and genome instability in cancer cells. Eur J Biochem. 1996 Jun 1;238(2):297–307. doi: 10.1111/j.1432-1033.1996.0297z.x. [DOI] [PubMed] [Google Scholar]
  48. Washburn B. K., Kushner S. R. Construction and analysis of deletions in the structural gene (uvrD) for DNA helicase II of Escherichia coli. J Bacteriol. 1991 Apr;173(8):2569–2575. doi: 10.1128/jb.173.8.2569-2575.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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