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. 1987 Feb;84(3):690–694. doi: 10.1073/pnas.84.3.690

3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

B B Konforti, R W Davis
PMCID: PMC304281  PMID: 3543934

Abstract

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  2. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cox M. M., Lehman I. R. Directionality and polarity in recA protein-promoted branch migration. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6018–6022. doi: 10.1073/pnas.78.10.6018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cox M. M., Lehman I. R. recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3433–3437. doi: 10.1073/pnas.78.6.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DasGupta C., Wu A. M., Kahn R., Cunningham R. P., Radding C. M. Concerted strand exchange and formation of Holliday structures by E. coli RecA protein. Cell. 1981 Aug;25(2):507–516. doi: 10.1016/0092-8674(81)90069-6. [DOI] [PubMed] [Google Scholar]
  6. Griffith J. D., Harris L. D., Register J., 3rd Visualization of SSB-ssDNA complexes active in the assembly of stable RecA-DNA filaments. Cold Spring Harb Symp Quant Biol. 1984;49:553–559. doi: 10.1101/sqb.1984.049.01.062. [DOI] [PubMed] [Google Scholar]
  7. Kahn R., Cunningham R. P., DasGupta C., Radding C. M. Polarity of heteroduplex formation promoted by Escherichia coli recA protein. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4786–4790. doi: 10.1073/pnas.78.8.4786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  10. Register J. C., 3rd, Griffith J. RecA protein filaments can juxtapose DNA ends: an activity that may reflect a function in DNA repair. Proc Natl Acad Sci U S A. 1986 Feb;83(3):624–628. doi: 10.1073/pnas.83.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Schnös M., Inman R. B. Position of branch points in replicating lambda DNA. J Mol Biol. 1970 Jul 14;51(1):61–73. doi: 10.1016/0022-2836(70)90270-6. [DOI] [PubMed] [Google Scholar]
  12. Shibata T., DasGupta C., Cunningham R. P., Williams J. G., Osber L., Radding C. M. Homologous pairing in genetic recombination. The pairing reaction catalyzed by Escherichia coli recA protein. J Biol Chem. 1981 Jul 25;256(14):7565–7572. [PubMed] [Google Scholar]
  13. Soltis D. A., Lehman I. R. recA protein promoted DNA strand exchange. J Biol Chem. 1983 May 25;258(10):6073–6077. [PubMed] [Google Scholar]
  14. West S. C., Cassuto E., Howard-Flanders P. Heteroduplex formation by recA protein: polarity of strand exchanges. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6149–6153. doi: 10.1073/pnas.78.10.6149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Wu A. M., Kahn R., DasGupta C., Radding C. M. Formation of nascent heteroduplex structures by RecA protein and DNA. Cell. 1982 Aug;30(1):37–44. doi: 10.1016/0092-8674(82)90009-5. [DOI] [PubMed] [Google Scholar]

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