Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity (original) (raw)
Summary
The activity of the _Eco_K DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage λ and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 103-to 104-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage λ. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the _Eco_K restriction/modification complex was tested and discounted because modification of λ was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in _Eco_K modification, was also discounted by the lack of any increase in the modification of λRal−, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.
Access this article
Subscribe and save
- Get 10 units per month
- Download Article/Chapter or eBook
- 1 Unit = 1 Article or 1 Chapter
- Cancel anytime Subscribe now
Buy Now
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Instant access to the full article PDF.
Similar content being viewed by others
References
- Appleyard RK (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39:440–452
Google Scholar - Bagg A, Kenyon CJ, Walker GC (1981) Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc Natl Acad Sci USA 78:5749–5753
Google Scholar - Battista JR, Ohta T, Nohmi T, Sun W, Walker GC (1990) Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis. Proc Natl Acad Sci USA 87:7190–7194
Google Scholar - Bertani G, Weigle JJ (1953) Host controlled variation in bacterial viruses. J Bacteriol 65:113–121
Google Scholar - Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Google Scholar - Blanco M, Herrera G, Collado P, Rebollo J, Botella LM (1982) Influence of RecA protein on induced mutagenesis. Biochimie 64:633–636
Google Scholar - Bolivar F, Rodriguez RC, Betlach MC, Boyer HW (1977) Construction and characterization of new cloning vehicles 1. Ampicillin-resistant derivatives of the plasmid pMB9. Gene 2:75–93
Google Scholar - Burckhardt SE, Woodgate R, Scheuermann RH, Echols H (1988) The UmuD mutagenesis protein of E. coli: over-production, purification and cleavage by RecA. Proc Natl Acad Sci USA 85:1811–1815
Google Scholar - Day RS (1977) UV-induced alleviation of K-specific restriction of bacteriophage λ. J Virol 21:1249–1251
Google Scholar - Dharmalingham K, Goldberg EB (1980) Restriction in vivo: V. Induction of SOS functions in Escherichia coli by restricted T4 phage DNA and alleviation of restriction by SOS functions. Mol Gen Genet 178:51–58
Google Scholar - Donnelly CE, Walker GC (1989) groE mutants of Escherichia coli are defective in _umuDC_-dependent UV mutagenesis. J Bacteriol 171:6117–6125
Google Scholar - Elledge SJ, Walker GC (1983) Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol 164:175–192
Google Scholar - Ennis DG, Ossana N, Mount DW (1989) Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage. J Bacteriol 171:2533–2541
Google Scholar - Freitag N, McEntee K (1989) “Activated”-RecA protein affinity chromatography of LexA repressor and other SOS-regulated proteins. Proc Natl Acad Sci USA 85:1806–1810
Google Scholar - Hiom KJ, Sedgwick SG (1991) Simple methods for making _Eco_K and McrA restriction mutants. Nucleic Acids Res 19:2502
Google Scholar - Howard-Flanders P, Simson E, Theriot L (1964) A locus that controls filament formation and sensitivity to radiation in Escherichia coli K-12. Genetics 49:237–241
Google Scholar - Iwasaki H, Shiba T, Nakata A, Shinagawa H (1988) Activation of UmuD protein for SOS mutagenesis by proteolytic processing mediated by RecA*. In: Freidberg EC, Hanawalt P (eds) Mechanisms and consequences of DNA damage processing. A. Liss, New York, pp 461–469
Google Scholar - Kato T, Shinoura Y (1977) Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol Gen Genet 156:121–131
Google Scholar - Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Google Scholar - Marsh L, Walker GC (1985) Cold sensitivity induced by overproduction of UmuDC in Escherichia coli. J Bacteriol 162:155–161
Google Scholar - Moreau P, Bailone A, Devoret R (1976) Prophage λ induction in Escherichia coli K-12 envA uvrB: a highly sensitive test for potential carcinogens. Proc Natl Acad Sci USA 73:3700–3704
Google Scholar - Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85:1816–1820
Google Scholar - Perry KL, Walker GC (1982) Identification of plasmid (pKM101)coded proteins involved in mutagenesis and UV resistance. Nature 300:278–281
Google Scholar - Sain B, Murray NE (1980) The hsd (host specificity) genes of _Eco_K K-12. Mol Gen Genet 180:35–46
Google Scholar - Sedgwick SG (1986) Inducible DNA repair in microbes. Microbiol Sci 3:76–83
Google Scholar - Sedgwick SG, Lodwick DL, Doyle N, Crowne HM, Strike P (1991) Functional complementation between chromosomal and plasmid mutagenic DNA repair genes. Mol Gen Genet 229:428–436
Google Scholar - Shinagawa H, Kato T, Ise T, Makino K, Nakata A (1983) Cloning and characterization of the umu operon responsible for inducible mutagenesis in Escherichia coli. Gene 23:167–174
Google Scholar - Shinagawa H, Iwasaki H, Kato T, Nakata A (1988) RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85:1806–1810
Google Scholar - Steinborn G (1978) Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. I. Isolation of uvm mutants and their phenotypical characterization in DNA repair and mutagenesis. Mol Gen Genet 165:87–93
Google Scholar - Studier FW, Bandyopadhyay PK (1988) Model for how type I restriction enzymes select cleavage sites in DNA. Proc Natl Acad Sci USA 85:4677–4681
Google Scholar - Thoms B, Wackernagel W (1982) UV-induced alleviation of λ restriction in Escherichia coli K-12: Kinetics of induction and specificity of this SOS function. Mol Gen Genet 186:111–117
Google Scholar - Thoms B, Wackernagel W (1984) Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA. Mol Gen Genet 197:297–303
Google Scholar - Toothman P (1981) Restriction alleviation by bacteriophages lambda and lambda reverse. J Virol 38:621–631
Google Scholar - Walker GC (1984) Mutagenesis and inducible responses to Deoxyribonucleic Acid damage in Escherichia coli. Microbiol Rev 48:60–93
Google Scholar - Witkin EM, McCall JO, Volkert MR, Wermundsen IE (1982) Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli. Mol Gen Genet 185:43–50
Google Scholar - Wood WB (1966) Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol 16:118–133
Google Scholar - Woodgate R, Rajagopalan M, Lu C, Echols H (1989) UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD′. Proc Natl Acad Sci USA 86:7301–7305
Google Scholar - Zabeau M, Friedman S, van Montagu M, Schell J (1980) The ral gene of phage λ. I. Identification of a non-essential gene that modulates restriction and modification in E. coli. Mol Gen Genet 179:63–73
Google Scholar
Author information
Authors and Affiliations
- Genetics Division, National Institute for Medical Research, NW7 1AA, Mill Hill, London, UK
Kevin J. Hiom & Steven G. Sedgwick
Authors
- Kevin J. Hiom
You can also search for this author inPubMed Google Scholar - Steven G. Sedgwick
You can also search for this author inPubMed Google Scholar
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Hiom, K.J., Sedgwick, S.G. Alleviation of _Eco_K DNA restriction in Escherichia coli and involvement of umuDC activity.Molec. Gen. Genet. 231, 265–275 (1992). https://doi.org/10.1007/BF00279800
- Received: 13 June 1991
- Issue Date: January 1992
- DOI: https://doi.org/10.1007/BF00279800