• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Apr 1989; 86(8): 2627–2631.
PMCID: PMC286970

Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli.

Abstract

The Escherichia coli recJ gene product was overproduced using a plasmid that carries the recJ gene downstream of a strong regulatable promoter and a strong ribosome-binding site. Overexpression of recJ produced a concomitant increase in the levels of single-stranded-DNA-specific nuclease activity present in crude cell extracts. This nuclease activity was purified to homogeneity and found to reside in a 60-kDa polypeptide. This polypeptide was induced with recJ overexpression and had the size and N-terminal amino acid sequence identical to the predicted RecJ protein sequence. The RecJ nuclease degraded linear single-stranded DNA but did not have exonuclease activity on linear double-stranded substrates or endonuclease activity on either single-stranded or double-stranded substrates. The RecJ exonuclease had greater activity on duplex DNA molecules with 5'-rather than 3'-single-stranded tails.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Horii Z, Clark AJ. Genetic analysis of the recF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J Mol Biol. 1973 Oct 25;80(2):327–344. [PubMed]
  • Lovett ST, Clark AJ. Genetic analysis of the recJ gene of Escherichia coli K-12. J Bacteriol. 1984 Jan;157(1):190–196. [PMC free article] [PubMed]
  • Kolodner R, Fishel RA, Howard M. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol. 1985 Sep;163(3):1060–1066. [PMC free article] [PubMed]
  • Lloyd RG, Picksley SM, Prescott C. Inducible expression of a gene specific to the RecF pathway for recombination in Escherichia coli K12. Mol Gen Genet. 1983;190(1):162–167. [PubMed]
  • Lloyd RG, Benson FE, Shurvinton CE. Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12. Mol Gen Genet. 1984;194(1-2):303–309. [PubMed]
  • Nakayama H, Nakayama K, Nakayama R, Irino N, Nakayama Y, Hanawalt PC. Isolation and genetic characterization of a thymineless death-resistant mutant of Escherichia coli K12: identification of a new mutation (recQ1) that blocks the RecF recombination pathway. Mol Gen Genet. 1984;195(3):474–480. [PubMed]
  • Amundsen SK, Taylor AF, Chaudhury AM, Smith GR. recD: the gene for an essential third subunit of exonuclease V. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5558–5562. [PMC free article] [PubMed]
  • Chaudhury AM, Smith GR. A new class of Escherichia coli recBC mutants: implications for the role of RecBC enzyme in homologous recombination. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7850–7854. [PMC free article] [PubMed]
  • Smith GR. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. [PMC free article] [PubMed]
  • Lovett ST, Luisi-DeLuca C, Kolodner RD. The genetic dependence of recombination in recD mutants of Escherichia coli. Genetics. 1988 Sep;120(1):37–45. [PMC free article] [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • Lovett ST, Clark AJ. Cloning of the Escherichia coli recJ chromosomal region and identification of its encoded proteins. J Bacteriol. 1985 Apr;162(1):280–285. [PMC free article] [PubMed]
  • Rogers SG, Weiss B. Exonuclease III of Escherichia coli K-12, an AP endonuclease. Methods Enzymol. 1980;65(1):201–211. [PubMed]
  • Hinkle DC, Chamberlin MJ. Studies of the binding of Escherichia coli RNA polymerase to DNA. I. The role of sigma subunit in site selection. J Mol Biol. 1972 Sep 28;70(2):157–185. [PubMed]
  • Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. [PubMed]
  • Kolodner R, Evans DH, Morrison PT. Purification and characterization of an activity from Saccharomyces cerevisiae that catalyzes homologous pairing and strand exchange. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5560–5564. [PMC free article] [PubMed]
  • Freifelder D, Dewitt R. Thermal hydrolysis as a means of opening supercoiled circles of DNA. Gene. 1977 Jul;1(5-6):385–387. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Sancar A, Wharton RP, Seltzer S, Kacinski BM, Clarke ND, Rupp WD. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. [PubMed]
  • LEHMAN IR, NUSSBAUM AL. THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. V. ON THE SPECIFICITY OF EXONUCLEASE I (PHOSPHODIESTERASE). J Biol Chem. 1964 Aug;239:2628–2636. [PubMed]
  • Chase JW, Richardson CC. Exonuclease VII of Escherichia coli. Purification and properties. J Biol Chem. 1974 Jul 25;249(14):4545–4552. [PubMed]
  • Kahn R, Cunningham RP, DasGupta C, Radding CM. Polarity of heteroduplex formation promoted by Escherichia coli recA protein. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4786–4790. [PMC free article] [PubMed]
  • Cox MM, Lehman IR. Directionality and polarity in recA protein-promoted branch migration. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6018–6022. [PMC free article] [PubMed]
  • Kushner SR, Nagaishi H, Templin A, Clark AJ. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. [PMC free article] [PubMed]
  • Kushner SR, Nagaishi H, Clark AJ. Indirect suppression of recB and recC mutations by exonuclease I deficiency. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1366–1370. [PMC free article] [PubMed]
  • Barbour SD, Nagaishi H, Templin A, Clark AJ. Biochemical and genetic studies of recombination proficiency in Escherichia coli. II. Rec+ revertants caused by indirect suppression of rec- mutations. Proc Natl Acad Sci U S A. 1970 Sep;67(1):128–135. [PMC free article] [PubMed]
  • Kushner SR, Nagaishi H, Clark AJ. Isolation of exonuclease VIII: the enzyme associated with sbcA indirect suppressor. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3593–3597. [PMC free article] [PubMed]
  • Joseph JW, Kolodner R. Exonuclease VIII of Escherichia coli. II. Mechanism of action. J Biol Chem. 1983 Sep 10;258(17):10418–10424. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • Gene
    Gene
    Gene links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • MedGen
    MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...