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. 1996 Jun;178(12):3550–3556. doi: 10.1128/jb.178.12.3550-3556.1996

In vivo stability of the Umu mutagenesis proteins: a major role for RecA.

E G Frank 1, M Gonzalez 1, D G Ennis 1, A S Levine 1, R Woodgate 1
PMCID: PMC178125  PMID: 8655553

Abstract

The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis. To avoid any gratuitous mutagenesis, the activity of the Umu proteins is normally kept to a minimum by tight transcriptional and posttranslational regulation. We have, however, previously observed that compared with an isogenic recA+ strain, the steady-state levels of the Umu proteins are elevated in a recA730 background (R. Woodgate and D. G. Ennis, Mol. Gen. Genet. 229:10-16, 1991). We have investigated this phenomenon further and find that another coprotease-constitutive (recA*) mutant, a recA432 strain, exhibits a similar phenotype. Analysis revealed that the increased steady-state levels of the Umu proteins in the recA* strains do indeed reflect an in vivo stabilization of the proteins. We have investigated the basis for the phenomenon and find that the mutant RecA* protein stabilizes the Umu proteins by not only converting the labile UmuD protein to the much more stable (and mutagenically active) UmuD' protein but by directly stabilizing UmuD' itself. In contrast, UmuC does not appear to be directly stabilized by RecA* but is instead dramatically stabilized in the presence of UmuD'. On the basis of these observations, we suggest that formation of a UmuD'C-RecA*-DNA quaternary complex protects the UmuD'C proteins from proteolytic degradation and as a consequence helps to promote the switch from error-free to error-prone mechanisms of DNA repair.

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Selected References

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  1. Bailone A., Sommer S., Knezević J., Dutreix M., Devoret R. A RecA protein mutant deficient in its interaction with the UmuDC complex. Biochimie. 1991 Apr;73(4):479–484. doi: 10.1016/0300-9084(91)90115-h. [DOI] [PubMed] [Google Scholar]
  2. Battista J. R., Ohta T., Nohmi T., Sun W., Walker G. C. Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7190–7194. doi: 10.1073/pnas.87.18.7190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bridges B. A., Woodgate R. Mutagenic repair in Escherichia coli: products of the recA gene and of the umuD and umuC genes act at different steps in UV-induced mutagenesis. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4193–4197. doi: 10.1073/pnas.82.12.4193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burckhardt S. E., Woodgate R., Scheuermann R. H., Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Craig N. L., Roberts J. W. E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide. Nature. 1980 Jan 3;283(5742):26–30. doi: 10.1038/283026a0. [DOI] [PubMed] [Google Scholar]
  6. Craig N. L., Roberts J. W. Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor. J Biol Chem. 1981 Aug 10;256(15):8039–8044. [PubMed] [Google Scholar]
  7. Donnelly C. E., Walker G. C. Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants. J Bacteriol. 1992 May;174(10):3133–3139. doi: 10.1128/jb.174.10.3133-3139.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Echols H., Goodman M. F. Mutation induced by DNA damage: a many protein affair. Mutat Res. 1990 Sep-Nov;236(2-3):301–311. doi: 10.1016/0921-8777(90)90013-u. [DOI] [PubMed] [Google Scholar]
  9. Ennis D. G., Fisher B., Edmiston S., Mount D. W. Dual role for Escherichia coli RecA protein in SOS mutagenesis. Proc Natl Acad Sci U S A. 1985 May;82(10):3325–3329. doi: 10.1073/pnas.82.10.3325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ennis D. G., Levine A. S., Koch W. H., Woodgate R. Analysis of recA mutants with altered SOS functions. Mutat Res. 1995 Jan;336(1):39–48. doi: 10.1016/0921-8777(94)00045-8. [DOI] [PubMed] [Google Scholar]
  11. Ennis D. G., Little J. W., Mount D. W. Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction. J Bacteriol. 1993 Nov;175(22):7373–7382. doi: 10.1128/jb.175.22.7373-7382.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ennis D. G., Ossanna N., Mount D. W. Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage. J Bacteriol. 1989 May;171(5):2533–2541. doi: 10.1128/jb.171.5.2533-2541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  14. Frank E. G., Hauser J., Levine A. S., Woodgate R. Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8169–8173. doi: 10.1073/pnas.90.17.8169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Freitag N., McEntee K. "Activated"-RecA protein affinity chromatography of LexA repressor and other SOS-regulated proteins. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8363–8367. doi: 10.1073/pnas.86.21.8363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Geuskens V., Mhammedi-Alaoui A., Desmet L., Toussaint A. Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease. EMBO J. 1992 Dec;11(13):5121–5127. doi: 10.1002/j.1460-2075.1992.tb05619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ho C., Kulaeva O. I., Levine A. S., Woodgate R. A rapid method for cloning mutagenic DNA repair genes: isolation of umu-complementing genes from multidrug resistance plasmids R391, R446b, and R471a. J Bacteriol. 1993 Sep;175(17):5411–5419. doi: 10.1128/jb.175.17.5411-5419.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hoyt M. A., Knight D. M., Das A., Miller H. I., Echols H. Control of phage lambda development by stability and synthesis of cII protein: role of the viral cIII and host hflA, himA and himD genes. Cell. 1982 Dec;31(3 Pt 2):565–573. doi: 10.1016/0092-8674(82)90312-9. [DOI] [PubMed] [Google Scholar]
  19. Joyce C. M., Grindley N. D. Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol. 1984 May;158(2):636–643. doi: 10.1128/jb.158.2.636-643.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kato T., Shinoura Y. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol Gen Genet. 1977 Nov 14;156(2):121–131. doi: 10.1007/BF00283484. [DOI] [PubMed] [Google Scholar]
  21. Kitagawa Y., Akaboshi E., Shinagawa H., Horii T., Ogawa H., Kato T. Structural analysis of the umu operon required for inducible mutagenesis in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4336–4340. doi: 10.1073/pnas.82.13.4336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lee M. H., Ohta T., Walker G. C. A monocysteine approach for probing the structure and interactions of the UmuD protein. J Bacteriol. 1994 Aug;176(16):4825–4837. doi: 10.1128/jb.176.16.4825-4837.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marsh L., Walker G. C. Cold sensitivity induced by overproduction of UmuDC in Escherichia coli. J Bacteriol. 1985 Apr;162(1):155–161. doi: 10.1128/jb.162.1.155-161.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murli S., Walker G. C. SOS mutagenesis. Curr Opin Genet Dev. 1993 Oct;3(5):719–725. doi: 10.1016/s0959-437x(05)80089-9. [DOI] [PubMed] [Google Scholar]
  25. Nohmi T., Battista J. R., Dodson L. A., Walker G. C. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1816–1820. doi: 10.1073/pnas.85.6.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peat T. S., Frank E. G., McDonald J. P., Levine A. S., Woodgate R., Hendrickson W. A. Structure of the UmuD' protein and its regulation in response to DNA damage. Nature. 1996 Apr 25;380(6576):727–730. doi: 10.1038/380727a0. [DOI] [PubMed] [Google Scholar]
  27. Petit M. A., Bedale W., Osipiuk J., Lu C., Rajagopalan M., McInerney P., Goodman M. F., Echols H. Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli. J Biol Chem. 1994 Sep 23;269(38):23824–23829. [PubMed] [Google Scholar]
  28. Rajagopalan M., Lu C., Woodgate R., O'Donnell M., Goodman M. F., Echols H. Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10777–10781. doi: 10.1073/pnas.89.22.10777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rebollo J. E., Moreau P. L., Blanco M., Devoret R. Restoration of RecA protein activity by genetic complementation. Mol Gen Genet. 1984;195(1-2):83–89. doi: 10.1007/BF00332728. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg M., Echols H. Differential recognition of ultraviolet lesions by RecA protein. Possible mechanism for preferential targeting of SOS mutagenesis to (6-4) dipyrimidine sites. J Biol Chem. 1990 Nov 25;265(33):20641–20645. [PubMed] [Google Scholar]
  31. Sassanfar M., Roberts J. W. Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication. J Mol Biol. 1990 Mar 5;212(1):79–96. doi: 10.1016/0022-2836(90)90306-7. [DOI] [PubMed] [Google Scholar]
  32. Shinagawa H., Iwasaki H., Kato T., Nakata A. RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1806–1810. doi: 10.1073/pnas.85.6.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sommer S., Bailone A., Devoret R. The appearance of the UmuD'C protein complex in Escherichia coli switches repair from homologous recombination to SOS mutagenesis. Mol Microbiol. 1993 Dec;10(5):963–971. doi: 10.1111/j.1365-2958.1993.tb00968.x. [DOI] [PubMed] [Google Scholar]
  34. Straus D., Walter W., Gross C. A. DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32. Genes Dev. 1990 Dec;4(12A):2202–2209. doi: 10.1101/gad.4.12a.2202. [DOI] [PubMed] [Google Scholar]
  35. Sweasy J. B., Witkin E. M., Sinha N., Roegner-Maniscalco V. RecA protein of Escherichia coli has a third essential role in SOS mutator activity. J Bacteriol. 1990 Jun;172(6):3030–3036. doi: 10.1128/jb.172.6.3030-3036.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Szekeres E. S., Jr, Woodgate R., Lawrence C. W. Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis. J Bacteriol. 1996 May;178(9):2559–2563. doi: 10.1128/jb.178.9.2559-2563.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tannock G. W., Luchansky J. B., Miller L., Connell H., Thode-Andersen S., Mercer A. A., Klaenhammer T. R. Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63. Plasmid. 1994 Jan;31(1):60–71. doi: 10.1006/plas.1994.1007. [DOI] [PubMed] [Google Scholar]
  38. Woodgate R. Construction of a umuDC operon substitution mutation in Escherichia coli. Mutat Res. 1992 Mar;281(3):221–225. doi: 10.1016/0165-7992(92)90012-7. [DOI] [PubMed] [Google Scholar]
  39. Woodgate R., Ennis D. G. Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage. Mol Gen Genet. 1991 Sep;229(1):10–16. doi: 10.1007/BF00264207. [DOI] [PubMed] [Google Scholar]
  40. Woodgate R., Rajagopalan M., Lu C., Echols H. UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7301–7305. doi: 10.1073/pnas.86.19.7301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Woodgate R., Sedgwick S. G. Mutagenesis induced by bacterial UmuDC proteins and their plasmid homologues. Mol Microbiol. 1992 Aug;6(16):2213–2218. doi: 10.1111/j.1365-2958.1992.tb01397.x. [DOI] [PubMed] [Google Scholar]
  42. Woodgate R., Singh M., Kulaeva O. I., Frank E. G., Levine A. S., Koch W. H. Isolation and characterization of novel plasmid-encoded umuC mutants. J Bacteriol. 1994 Aug;176(16):5011–5021. doi: 10.1128/jb.176.16.5011-5021.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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