Skip to main content
SpringerLink
Log in

DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: Effects of mutations and treatments that alter levels of exonuclease V or RecA protein

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

Certain strains suppress the temperature-sensitivity caused by ssb-1, which encodes a mutant ssDNA binding protein (SSB). At 42°C, such strains are extremely UV-sensitive, degrade their DNA extensively after UV irradiation, and are deficient in UV mutability and UV induction of recA protein synthesis. We transduced recC22, which eliminates Exonuclease V activity, and recAo281, which causes operator-constitutive synthesis of recA protein, into such an ssb-1 strain. Both double mutants degraded their DNA extensively at 42°C after UV irradiation, and both were even more UV-sensitive than the ssb-1 single mutant. We conclude that one or more nucleases other than Exonuclease V degrades DNA in the ssb recC strain, and that recA protein, even if synthesized copiously, can function efficiently in recombinational DNA repair and in control of post-UV DNA degradation only if normal SSB is also present. Pretreatment with nalidixic acid at 30°C restored normal UV mutability at 42°C, but did not increase UV resistance, in an ssb-1 strain. Another ssb allele, ssb-113, which blocks SOS induction at 30°C, increases spontaneous mutability more than tenfold. The ssb-113 allele was transduced into the SOS-constitutive recA730 strain SC30. This double mutant expressed the same elevated spontaneous and UV-induced mutability at 30°C as the ssb + recA730 strain, and was three times more UV-resistant than its ssb-113 recA +parent. We conclude that ssb-1 at 42°C and ssb-113 at 30°C block UV-induced activation of recA protease, but that neither allele interferes with subsequent steps in SOS-mediated mutagenesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Referenes

  • Baluch J, Chase JW, Sussman R (1980) Synthesis of recA protein and induction of bacteriophage lambda in single-strand deoxyribonucleic acid-binding protein mutants of Escherichia coli. J Bacteriol 144:489–498

    Google Scholar 

  • Cassuto E, West SC, Mursalim J, Conlon S, Howard-Flanders P (1980) Initiation of genetic recombination: homologous pairing between duplex DNA molecules promoted by recA protein. Proc Natl Acad Sci USA 77:3962–3966

    Google Scholar 

  • Chrysogelos S, Griffith J (1982) Escherichia coli single-strand binding protein organizes single-stranded DNA in nucleosome-like units. Proc Natl Acad Sci USA 79:5803–5807

    Google Scholar 

  • Clarke CH, Hill RF (1972) Mutation frequency decline for streptomycin-resistant mutations induced by ultraviolet light in Escherichia coli B/r. Mutat Res 14:247–249

    Google Scholar 

  • Cox MM, Lehman IR (1981a) recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci USA 78:3433–3437

    Google Scholar 

  • Cox MM, Lehman IR (1981b) Directionality and polarity in recA protein-promoted branch migration. Proc Natl Acad Sci USA 78:6018–6022

    Google Scholar 

  • Craig NL, Roberts JW (1980) E. coli recA protein-directed cleavage of phage λ repressor requires polynucleotide. Nature 283:26–30

    Google Scholar 

  • Cunningham RP, DasGupta C, Shibata T, Radding CM (1980) Homologous pairing in genetic recombination: recA protein makes joint molecules of gapped circular DNA and closed circular DNA. Cell 20:223–235

    Google Scholar 

  • DasGupta C, Radding CM (1982) Polar branch migration promoted by recA protein: effect of mismatched base pairs. Proc Natl Acad Sci USA 79:762–766

    Google Scholar 

  • DasGupta C, Shibata T, Cunningham RP, Radding CM (1980) The topology of homologous pairing promoted by recA protein. Cell 22:437–446

    Google Scholar 

  • DasGupta C, Wu AM, Kahn R, Cunningham RP, Radding CM (1981) Concerted strand exchange and formation of holliday structures by E. coli recA protein. Cell 25:507–516

    Google Scholar 

  • Emmerson PT (1968) Recombination deficient mutants of Escherichia coli K12 that map between thyA and argA. Genetics 60:19–30

    Google Scholar 

  • Flory J, Radding CM (1982) Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein. Cell 28:747–756

    Google Scholar 

  • Glassberg J, Meyer RR, Kornberg A (1979) Mutant single-strand binding protein of Escherichia coli: genetic and physiological characterization. J Bacteriol 140:14–19

    Google Scholar 

  • Greenberg J, Berends L, Donch J, Johnson B (1975) Reversion studies with exrB in Escherichia coli. Genet Res Camb 25:109–117

    Google Scholar 

  • Johnson BF (1977) Genetic mapping of the lexC-113 mutation. Mol Gen Genet 157:91–97

    Google Scholar 

  • Kahn R, Cunningham RP, DasGupta C, Radding CM (1981) Polarity of heteroduplex formation promoted by Escherichia coli recA protein. Proc Natl Acad Sci USA 78:4786–4790

    Google Scholar 

  • Kunkel TA, Meyer RR, Loeb LA (1979) Single-strand binding protein enhances fidelity of DNA synthesis in vitro. Proc Natl Acad Sci USA 76:6331–6335

    Google Scholar 

  • Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80:575–599

    Google Scholar 

  • Lieberman HB, Witkin EM (1981) Variable expression of the ssb-1 allele in different strains of Escherichia coli K12 and B: differential suppression of its effects on DNA replication, DNA repair and ultraviolet mutagenesis. Mol Gen Genet 183:348–355

    Google Scholar 

  • Little JW, Mount DW (1982) The SOS regulatory system of Escherichia coli. Cell 29:11–22

    Google Scholar 

  • Livneh Z, Lehman IR (1982) Recombinational bypass of pyrimidine dimers promoted by the recA protein of Escherichia coli. Proc Natl Acad Sci USA 79:3171–3175

    Google Scholar 

  • MacKay V, Linn S (1974) The mechanism of degradation of duplex deoxyribonucleic acid by the recBC enzyme of Escherichia coli K-12. J Biol Chem 249:4286–4294

    Google Scholar 

  • Marsden HS, Pollard EC, Ginoza W, Randall EP (1974) Involvement of recA and exr genes in the in vivo inhibition of the recBC nuclease. J Bacteriol 118:465–470

    Google Scholar 

  • McEntee K, Weinstock GM, Lehman IR (1979) Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc Natl Acad sci USA 76:2615–2619

    Google Scholar 

  • McEntee K, Weinstock GM, Lehman IR (1980) recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein. Proc Natl Acad Sci USA 77:857–861

    Google Scholar 

  • Meyer RR, Voegele DW, Ruben SM, Rein DC, Trela JM (1982) Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli. Mutat Res 94:299–313

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Ogawa T, Wabiko H, Tsurimoto T, Horii T, Masukata H, Ogawa H (1979) Characteristics of purified recA protein and the regulation of its synthesis in vivo. Cold Spring Harbor Symp Quant Biol 43:909–915

    Google Scholar 

  • Phizicky EM, Roberts JW (1981) Induction of SOS function: regulation of proteolytic activity of E. coli recA protein by interaction with DNA and nucleoside triphosphate. Cell 25:259–267

    Google Scholar 

  • Prell A, Wackernagel W (1981) Effect of recA protein on the DNase activities of the recBC enzyme. J Biol Chem 256:10415–10419

    Google Scholar 

  • Resnick J, Sussman R (1982) Escherichia coli single-strand DNA binding protein from wild type and lexC113 mutant affects in vitro proteolytic cleavage of phage λ repressor. Proc Natl Acad Sci USA 79:2832–2835

    Google Scholar 

  • Roberts JW, Roberts CW, Craig NL, Phizicky EM (1979) Activity of the Escherichia coli recA-gene product. Cold Spring Harbor Symp Quant Biol 43:917–920

    Google Scholar 

  • Sevastopoulos CG, Wehr CT, Glaser DA (1977) Large-scale automated isolation of Escherichia coli mutants with thermosensitive DNA replication. Proc Natl Acad Sci USA 74:3485–3489

    Google Scholar 

  • Shibata T, DasGupta C, Cunningham RP, Radding CM (1979) Purified E. coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. Proc Natl Acad Sci USA 76:1638–1642

    Google Scholar 

  • Vales LD, Chase JW, Murphy JB (1980) Effect of the ssbA1 and lexC113 mutations on lambda prophage induction, phage growth and cell survival. J Bacteriol 143:887–896

    Google Scholar 

  • Vogel HJ, Bonner DM (1956) Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 218:97–106

    Google Scholar 

  • Volkert MR, George DL, Witkin EM (1976) Partial suppression of the lexA phenotype by mutations (rum) which restore ultraviolet resistance but not ultraviolet mutability to Escherichia coli B/r uvrA lexA. Mutat Res 36:17–28

    Google Scholar 

  • Volkert MR, Margossian LJ, Clark AJ (1981) Evidence that rnmB is the operator of the Escherichia coli recA gene. Proc Natl Acad Sci USA 78:1786–1790

    Google Scholar 

  • Volkert MR, Spencer DF, Clark AJ (1979) Indirect and intragenic suppression of the lexA102 mutation in E. coli B/r. Mol Gen Genet 117:129–137

    Google Scholar 

  • Wang TV, Smith KC (1982) Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated δuvrB strains of Escherichia coli K-12. J Bacteriol 151:186–192

    Google Scholar 

  • Weinstock GM, McEntee K (1981) RecA protein-dependent proteolysis of bacteriophage λ repressor. Characterization of the reaction and stimulation by DNA-binding proteins. J Biol Chem 256:10883–10888

    Google Scholar 

  • Weinstock GM, McEntee K, Lehman IR (1979) ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli. Proc Natl Acad Sci USA 76:126–130

    Google Scholar 

  • West SC, Cassuto E, Howard-Flanders P (1981) recA protein promotes homologous-pairing and strand-exchange reactions between duplex DNA molecules. Proc Natl Acad Sci USA 78:2100–2104

    Google Scholar 

  • West SC, Cassuto E, Howard-Flanders P (1982) Role of SSB protein in recA promoted branch migration reactions. Mol Gen Genet 186:333–338

    Google Scholar 

  • West SC, Emmerson PT (1977) Induction of protein synthesis in Escherichia coli following UV- or γ-irradiation, mitomycin C treatment or tif expression. Mol Gen Genet 151:57–67

    Google Scholar 

  • Whittier RF, Chase JW (1981) DNA repair in E. coli strains deficient in single-strand DNA binding protein. Mol Gen Genet 183:341–347

    Google Scholar 

  • Whittier RF, Chase JW (1983) DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein. Mol Gen Genet 190:101–111

    Google Scholar 

  • Willetts NS, Clark AJ (1969) Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol 100:231–239

    Google Scholar 

  • Witkin EM (1974) Thermal enhancement of ultraviolet mutability in a tif-1 uvr A derivative of Escherichia coli B/r: evidence that ultraviolet mutagenesis depends upon an inducible function. Proc Natl Acad Sci USA 71:1930–1934

    Google Scholar 

  • Witkin EM (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev 40:869–907

    Google Scholar 

  • Witkin EM (1982) From Gainsville to Toulouse: the evolution of a model. Biochimie 64:549–555

    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 Genet 185:43–50

    Google Scholar 

Download references

Author information

Author notes

  1. Howard B. Lieberman

    Present address: Department of Biology, Kline Biology Tower, Yale University, 06511, New Haven, Connecticut, USA

Authors and Affiliations

  1. Department of Biological Sciences, Rutgers University, Douglass Campus, 08903, New Brunswick, New Jersey, USA

    Howard B. Lieberman & Evelyn M. Witkin

Authors
  1. Howard B. Lieberman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Evelyn M. Witkin
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by B.A. Bridges

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lieberman, H.B., Witkin, E.M. DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: Effects of mutations and treatments that alter levels of exonuclease V or RecA protein. Mol Gen Genet 190, 92–100 (1983). https://doi.org/10.1007/BF00330329

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00330329

Keywords

Search

Navigation