Biochemistry (Moscow)

, Volume 83, Issue 3, pp 281–293 | Cite as

Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System

  • M. V. Monakhova
  • A. I. Penkina
  • A. V. Pavlova
  • A. M. Lyaschuk
  • V. V. Kucherenko
  • A. V. Alexeevski
  • V. G. Lunin
  • P. Friedhoff
  • G. Klug
  • T. S. Oretskaya
  • E. A. KubarevaEmail author


We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C–terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N–terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.


DNA repair mismatch MutL protein 



amino acid; bp


base pair




MutL from Escherichia coli


Gyrase Hsp90, Histidine Kinase, MutL family of ATPases


isopropyl β–D–1–thiogalactopyranoside


lysogeny broth


mismatch repair system


phenylmethylsulfonyl fluoride


MutL from Rhodobacter sphaeroides


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Guarne, A. (2012) The functions of MutL in mismatch repair: the power of multitasking, Prog. Mol. Biol. Transl. Sci., 110, 41–70.CrossRefPubMedGoogle Scholar
  2. 2.
    Iyer, R. R., Pluciennik, A., Burdett, V., and Modrich, P. L. (2006) DNA mismatch repair: functions and mechanisms, Chem. Rev., 106, 302–323.CrossRefPubMedGoogle Scholar
  3. 3.
    Jiricny, J. (2013) Postreplicative mismatch repair, Cold Spring Harb. Perspect. Biol., 5, a012633.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lynch, H. T., Lynch, P. M., Lanspa, S. J., Snyder, C. L., Lynch, J. F., and Boland, C. R. (2009) Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications, Clin. Genet., 76, 1–18.PubMedGoogle Scholar
  5. 5.
    Michailidi, C., Papavassiliou, A. G., and Troungos, C. (2012) DNA repair mechanisms in colorectal carcinogenesis, Curr. Mol. Med., 12, 237–246.CrossRefPubMedGoogle Scholar
  6. 6.
    Lahue, R. S., and Modrich, P. (1988) Methyldirected DNA mismatch repair in Escherichia coli, Mutat. Res., 198, 37–43.CrossRefPubMedGoogle Scholar
  7. 7.
    Längle–Rouault, F., Maenhaut–Michel, G., and Radman, M. (1987) GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair, EMBO J., 6, 1121–1127.PubMedPubMedCentralGoogle Scholar
  8. 8.
    Modrich, P. (1989) Methyldirected DNA mismatch correction, J. Biol. Chem., 264, 6597–6600.PubMedGoogle Scholar
  9. 9.
    Perevozchikova, S. A., Romanova, E. A., Oretskaya, T. S., Friedhoff, P., and Kubareva, E. A. (2013) Modern concepts of the structural and functional organization of the system for the DNA noncanonical nucleotide pair repair, Acta Naturae, 5, 27–44.Google Scholar
  10. 10.
    Hall, M. C., Shcherbakova, P. V., and Kunkel, T. A. (2002) Differential ATP binding and intrinsic ATP hydrolysis by aminoterminal domains of the yeast Mlh1 and Pms1 proteins, J. Biol. Chem., 277, 3673–3679.CrossRefPubMedGoogle Scholar
  11. 11.
    Sancar, A., and Hearst, J. E. (1993) Molecular matchmakers, Science, 259, 1415–1420.CrossRefPubMedGoogle Scholar
  12. 12.
    Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Endonucleolytic function of MutLa in human mismatch repair, Cell, 126, 297–308.CrossRefPubMedGoogle Scholar
  13. 13.
    Kadyrov, F. A., Holmes, S. F., Arana, M. E., Lukianova, O. A., O’Donnell, M., Kunkel, T. A., and Modrich, P. (2007) Saccharomyces cerevisiae MutLa is a mismatch repair endonuclease, J. Biol. Chem., 282, 37181–37190.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kosinski, J., Plotz, G., Guarne, A., Bujnicki, J. M., and Friedhoff, P. (2008) The PMS2 subunit of human MutLa contains a metal ion binding domain of the iron–dependent repressor protein family, J. Mol. Biol., 382, 610–627.CrossRefPubMedGoogle Scholar
  15. 15.
    Correa, E. M. E., Martina, M. A., De Tullio, L., Argaran, C. E., and Barra, J. L. (2011) Some amino acids of the Pseudomonas aeruginosa MutL D(Q/M)HA(X)(2)E(X)(4)E conserved motif are essential for the in vivo function of the protein but not for the in vitro endonuclease activity, DNA Repair, 10, 1106–1113.CrossRefPubMedGoogle Scholar
  16. 16.
    Duppatla, V., Bodda, C., Urbanke, C., Friedhoff, P., and Rao, D. N. (2009) The carboxyterminal domain is sufficient for endonuclease activity of Neisseria gonorrhoeae MutL, Biochem. J., 423, 265–277.CrossRefPubMedGoogle Scholar
  17. 17.
    Fukui, K., Nishida, M., Nakagawa, N., Masui, R., and Kuramitsu, S. (2008) Bound nucleotide controls the endonuclease activity of mismatch repair enzyme MutL, J. Biol. Chem., 283, 12136–12145.CrossRefPubMedGoogle Scholar
  18. 18.
    Iino, H., Kim, K., Shimada, A., Masui, R., Kuramitsu, S., and Fukui, K. (2011) Characterization of C–and N–terminal domains of Aquifex aeolicus MutL endonuclease: N–terminal domain stimulates the endonuclease activity of C–terminal domain in a zinc–dependent manner, Biosci. Rep., 31, 309–322.CrossRefPubMedGoogle Scholar
  19. 19.
    Pillon, M. C., Lorenowicz, J. J., Uckelmann, M., Klocko, A. D., Mitchell, R. R., Chung, Y. S., Modrich, P., Walker, G. C., Simmons, L. A., Friedhoff, P., and Guarne, A. (2010) Structure of the endonuclease domain of MutL: unlicensed to cut, Mol. Cell, 39, 145–151.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bijlsma, J. J., and Groisman, E. A. (2003) Making informed decisions: regulatory interactions between two–component systems, Trends Microbiol., 11, 359–366.CrossRefPubMedGoogle Scholar
  21. 21.
    Capra, E. J., and Laub, M. T. (2012) Evolution of two–component signal transduction systems, Annu. Rev. Microbiol., 66, 325–347.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Banasik, M., and Sachadyn, P. (2014) Conserved motifs of MutL proteins, Mutat. Res., 769, 69–79.CrossRefPubMedGoogle Scholar
  23. 23.
    Ban, C., and Yang, W. (1998) Crystal structure and ATPase activity of MutL: implications for DNA repair and mutagenesis, Cell, 95, 541–552.CrossRefPubMedGoogle Scholar
  24. 24.
    Dutta, R., and Inouye, M. (2000) GHKL, an emergent ATPase/kinase superfamily, Trends Biochem. Sci., 25, 24–28.CrossRefPubMedGoogle Scholar
  25. 25.
    Plotz, G., Raedle, J., Brieger, A., Trojan, J., and Zeuzem, S. (2003) N–terminus of hMLH1 confers interaction of hMutLa and hMutLβ with hMutSa, Nucleic Acids Res., 31, 3217–3226.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pillon, M. C., Miller, J. H., and Guarne, A. (2011) The endonuclease domain of MutL interacts with the beta sliding clamp, DNA Repair (Amst.), 10, 87–93.CrossRefGoogle Scholar
  27. 27.
    Davis, R. W., Botstein, D., and Roth, J. R. (1980) Advanced Bacterial Genetics: a Manual for Genetic Engineering, Cold Spring Harbor, Cold Spring Harbor Laboratory, N.Y.Google Scholar
  28. 28.
    Winkler, I., Marx, A. D., Lariviere, D., Heinze, R. J., Cristovao, M., Reumer, A., Curth, U., Sixma, T. K., and Friedhoff, P. (2011) Chemical trapping of the dynamic MutS–MutL complex formed in DNA mismatch repair in Escherichia coli, J. Biol. Chem., 286, 17326–17337.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Echave, J., Spielman, S. J., and Wilke, C. O. (2016) Causes of evolutionary rate variation among protein sites, Nat. Rev. Genet., 17, 109–121.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Namadurai, S., Jain, D., Kulkarni, D. S., Tabib, C. R., Friedhoff, P., Rao, D. N., and Nair, D. T. (2010) The C–terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer, PloS One, 5, 13726.CrossRefGoogle Scholar
  31. 31.
    Jacquelin, D. K., Filiberti, A., Argara?a, C. E., and Barra, J. L. (2005) Pseudomonas aeruginosa MutL protein functions in Escherichia coli, Biochem. J., 388, 879–887.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685.CrossRefPubMedGoogle Scholar
  33. 33.
    Carson, M., Johnson, D. H., McDonald, H., Brouillette, C., and Delucas, L. J. (2007) His–tag impact on structure, Acta Crystallogr. D. Biol. Crystallogr., 63, 295–301.CrossRefPubMedGoogle Scholar
  34. 34.
    Choudhary, M., Mackenzie, C., Nereng, K. S., Sodergren, E., Weinstock, G. M., and Kaplan, S. (1994) Multiple chromosomes in bacteria: structure and function of chromosome II of Rhodobacter sphaeroides 2.4.1, J. Bacteriol., 176, 7694–7702.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Ban, C., Junop, M., and Yang, W. (1999) Transformation of MutL by ATP binding and hydrolysis: a switch in DNA mismatch repair, Cell, 97, 85–97.CrossRefPubMedGoogle Scholar
  36. 36.
    Lakhin, A. V., Efremova, A. S., Makarova, I. V., Grishina, E. E., Shram, S. I., Tarantul, V. Z., and Gening, L. V. (2013) Effect of Mn(II) on the errorprone DNA polymerase iota activity in extracts from human normal and tumor cells, Mol. Genet. Mikrobiol. Virusol., 1, 14–20.Google Scholar
  37. 37.
    Frank, E. G., and Woodgate, R. (2007) Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese, J. Biol. Chem., 282, 24689–24696.CrossRefPubMedGoogle Scholar
  38. 38.
    Zakharcheva, K. A., Gening, L. V., Kazachenko, K. Yu., and Tarantul, V. Z. (2017) Cells resistant to toxic concentrations of manganese have increased ability to repair DNA, Biochemistry (Moscow), 82, 101–110.CrossRefGoogle Scholar
  39. 39.
    Grilley, M., Welsh, K. M., Su, S. S., and Modrich, P. (1989) Isolation and characterization of the Escherichia coli mutL gene product, J. Biol. Chem., 264, 1000–1004.PubMedGoogle Scholar
  40. 40.
    Prolla, T. A., Pang, Q., Alani, E., Kolodner, R. D., and Liskay, R. M. (1994) MLH1, PMS1, and MSH2 interactions during the initiation of DNA mismatch repair in yeast, Science, 265, 1091–1093.PubMedGoogle Scholar
  41. 41.
    Drotschmann, K., Aronshtam, A., Fritz, H. J., and Marinus, M. G. (1998) The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA, Nucleic Acids Res., 26, 948–953.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Acharya, S., Foster, P. L., Brooks, P., and Fishel, R. (2003) The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair, Mol. Cell, 12, 233–246.CrossRefPubMedGoogle Scholar
  43. 43.
    Mordasini, T., Curioni, A., and Andreoni, W. (2003) Why do divalent metal ions either promote or inhibit enzymatic reactions? The case of BamHI restriction endonuclease from combined quantum–classical simulations, J. Biol. Chem., 278, 4381–4384.CrossRefPubMedGoogle Scholar
  44. 44.
    Pingoud, A., Fuxreiter, M., Pingoud, V., and Wende, W. (2005) Type II restriction endonucleases: structure and mechanism, Cell Mol. Life Sci., 62, 685–707.CrossRefPubMedGoogle Scholar
  45. 45.
    Mauris, J., and Evans, T. C. (2009) Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity, PLoS One, 4, 71–75.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • M. V. Monakhova
    • 1
  • A. I. Penkina
    • 2
  • A. V. Pavlova
    • 2
  • A. M. Lyaschuk
    • 3
  • V. V. Kucherenko
    • 4
  • A. V. Alexeevski
    • 1
    • 5
  • V. G. Lunin
    • 3
  • P. Friedhoff
    • 6
  • G. Klug
    • 7
  • T. S. Oretskaya
    • 2
  • E. A. Kubareva
    • 1
    Email author
  1. 1.Belozersky Institute of Physico–Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Chemistry DepartmentLomonosov Moscow State UniversityMoscowRussia
  3. 3.Gamaleya Research Institute of Epidemiology and MicrobiologyMoscowRussia
  4. 4.Bioengineering and Bioinformatics DepartmentLomonosov Moscow State UniversityMoscowRussia
  5. 5.Research Institute of System DevelopmentMoscowRussia
  6. 6.Institut für BiochemieJustus–Liebig–UniversitätGieβenGermany
  7. 7.Institut für Mikrobiologie und MolekularbiologieJustus–Liebig–UniversitätGieβenGermany

Personalised recommendations