Skip to main content
SpringerLink
Log in

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments

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

Summary

The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.

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

Abbreviations

RecAwt:

RecA wind-type protein

ssDNA:

singlestranded DNA

dsDNA:

dmble-stranded DNA

References

  • Bailone A, Levine A, Devoret R (1979) In vivo inactivation of prophage λ repressor. J Mol Biol 131:553–572

    Google Scholar 

  • Bailone A, Bäckman A, Sommer S, Célérier J, Bagdasarian MM, Bagdasarian M, Devoret R (1988) PsiB polypeptide prevents activation of RecA protein in Escherichia coli. Mol Gen Genet 214:389–395

    Google Scholar 

  • Bailone A, Sommer S, Knezevic J, Dutreix M, Devoret R (1991) A RecA protein mutant deficient in its interaction with the UmuDC complex. Biochimie 73:479–484

    Google Scholar 

  • Brenner SL, Zlotnick A, Griffith JD (1988) RecA protein selfassembly multiple discrete aggregation states. J Mol Biol 204:959–972

    Google Scholar 

  • Burckhardt SE, Woodgate R, Scheuermann RH, Echols H (1988) UmuD mutagenesis protein of Escherichia coli: Overproduction, purification and cleavage by RecA. Proc Natl Acad Sci USA 85:1811–1815

    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 

  • Clark AJ (1973) Recombination deficient mutants of E. coli and other bacteria. Annu Rev Genet 7:67–86

    Google Scholar 

  • Connolly B, West SC (1990) Genetic recombination in Escherichia coli: Holliday junctions made by RecA protein are resolved by fractionated cell-free extracts. Proc Natl Acad Sci USA 87:8476–8480

    Google Scholar 

  • Cox MM, McEntee K, Lehman IR (1981) A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli. J Biol Chem 256:4676–4678

    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, 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 

  • Devoret R, Pierre M, Moreau PL (1983) Prophage ϕ80 is induced in Escherichia coli K12 recA430. Mol Gen Genet 189:199–206

    Google Scholar 

  • Devoret R, Bailone A, Dutreix M, Moreau PL, Sommer S, Bagdasarian M (1988) Regulation of activation of RecA protein in E. coli. In: Friedberg EC, Hanawalt PC (eds) UCLA symposia on molecular and cellular biology: Mechanisms and consequences of DNA damage processing. Alan R. Liss, New York, pp 437–443

    Google Scholar 

  • Dutreix M, Bailone A, Devoret R (1985) Efficiency of induction of prophage λ mutants as a function of recA alleles. J Bacteriol 161:1080–1085

    Google Scholar 

  • Dutreix M, Moreau PL, Bailone A, Galibert F, Battista JR, Walker GC, Devoret R (1989) New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UVmutagenesis. J Bacteriol 171:2415–2423

    Google Scholar 

  • Ginsburg H, Edmiston SH, Harper J, Mount DW (1982) Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K12. Mol Gen Genet 187:4–11

    Google Scholar 

  • Heuser J, Griffith J (1989) Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy. J Mol Biol 210:473–484

    Google Scholar 

  • Konrad EB (1977) Method for the isolation of Escherichia coli mutants with enhanced recombination between chromosomal duplications. J Bacteriol 130:167–172

    Google Scholar 

  • Kowalczykowski SC, Burk DL, Krupp RA (1989) Biochemical events essential to the recombination activity of Escherichia coli RecA protein. I. Properties of the mutant RecA142 protein. J Mol Biol 207:719–723

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Google Scholar 

  • Lavery PE, Kowalczykowski SC (1988) Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli. J Mol Biol 203:861–864

    Google Scholar 

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

    Google Scholar 

  • Little JW, Edmiston SH, Pacelli LZ, Mount DW (1980) Cleavage of the Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci USA 77:3225–3229

    Google Scholar 

  • Menetski JP, Bear DG, Kowalczykowski SC (1990) Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis. Proc Natl Acad Sci USA 87:21–25

    Google Scholar 

  • Moreau PL (1987) Effects of overproduction of single-stranded DNA binding protein on RecA-protein-dependent processes in Escherichia coli. J Mol Biol 194:621–634

    Google Scholar 

  • Moreau PL, Pélico JV, Devoret R (1982) Cleavage of λ repressor and synthesis of RecA protein induced by transferred UVdamaged F sex factor. Mol Gen Genet 186:170–179

    Google Scholar 

  • Nohmi T, Battista JR, Dodson LA, Walker GC (1988) RecAmediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci USA 85:1816–1820

    Google Scholar 

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

    Google Scholar 

  • Quillardet P, Moreau PL, Ginsburg H, Mount DW, Devoret R (1982) Cell survival, UV-reactivation and induction of prophage λ in Escherichia coli K 12 overproducing RecA protein. Mol Gen Genet 188:37–43

    Google Scholar 

  • Radding CM (1988) Homologous pairing and strand exchange promoted by Escherichia coli RecA protein. In: Kucherlapati R, Smith GR (eds) Genetic recombination. American Society for Microbiology Washington, DC, pp 193–229

    Google Scholar 

  • Roberts JW, Roberts CW (1981) Two mutations that alter the regulatory activity of E. coli RecA protein. Nature 290: 422–242

    Google Scholar 

  • Roberts JW, Roberts CW, Mount DW (1977) Inactivation and proteolytic cleavage of phage λ repressor in vitro in an ATPdependent reaction. Proc Natl Acad Sci USA 74:2283–2287

    Google Scholar 

  • Roca AI, Cox MM (1990) The RecA protein: Structure and function. Crit Rev Biochem Mol Biol 25:415–456

    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 

  • Shibata T, DasGupta C, Cunningham RP, Williams JG, Osber L, Radding CM (1981) Homologous pairing in genetic recombination. The pairing reaction catalyzed by E. coli RecA protein J Biol Chem 256:7565–7572

    Google Scholar 

  • Shinagawa H, Iwasaki H, Kato T, Nakata A (1988) RecA proteindependent cleavage of UmuD protein and SOS mutagenesis. Proc Natl Acad Sci USA 85:1806–1810

    Google Scholar 

  • Stasiak A, Egelman EH (1988) Visualization of recombination reactions. In: Kucherlapati R, Smith GR (eds) Genetic recombination, chapter 8. American Society for Microbiology Washington, pp 265–307

  • Story RM, Weber IT, Steitz TA (1992) The structure of the E. coli recA protein monomer and polymer. Nature 355:318–325

    Google Scholar 

  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89

    Google Scholar 

  • Takahashi M, Kubista M, Norden B (1991) Co-ordination of multiple DNA molecules in RecA fiber evidenced by linear dichroism spectroscopy. Biochimie 73:219–227

    Google Scholar 

  • Umezu K, Nakayama K, Nakayama H (1990) Escherichia coli RecQ protein is a DNA helicase. Proc Natl Acad Sci USA 87:5363–5367

    Google Scholar 

  • Walker GC (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48:60–93

    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 

Download references

Author information

Authors and Affiliations

  1. Groupe d'Etude “Mutagénèse et Cancerogénèse”, Laboratoire d'Enzymologie, CNRS, F-91198, Gif-sur-Yvette, France

    Marie Dutreix, Adriana Bailone & Raymond Devoret

  2. The Departments of Genetics and Molecular Biophysics & Biochemistry, Yale University School of Medicine New Haven, 06510, CT, USA

    Bruce Burnett & Charles M. Radding

Authors
  1. Marie Dutreix
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruce Burnett
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adriana Bailone
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles M. Radding
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Raymond Devoret
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by B. Kilbey

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dutreix, M., Burnett, B., Bailone, A. et al. A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments. Molec. Gen. Genet. 232, 489–497 (1992). https://doi.org/10.1007/BF00266254

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00266254

Key words

Search

Navigation