Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Aug;85(16):5854–5858. doi: 10.1073/pnas.85.16.5854

Sensitive homologous recombination strand-transfer assay: partial purification of a Drosophila melanogaster enzyme and detection of sequence effects on the strand-transfer activity of RecA protein.

J G McCarthy 1, M Sander 1, K Lowenhaupt 1, A Rich 1
PMCID: PMC281863  PMID: 3137561

Abstract

A sensitive homologous recombination strand-transfer assay is described that employs short radiolabeled double-stranded DNA fragments from the lac/polylinker region of plasmid pUC18 and (+)viral M13mp18 single-stranded DNA as substrates. Substitution of a short radiolabeled double-stranded fragment for full-length linear M13 double-stranded DNA results in an assay whose sensitivity is improved greater than 8-fold. In addition, it is less sensitive to interference from nucleases or ligases than previous assays. The assay was used to partially purify an ATP-independent strand-transfer activity from a crude nuclear extract of Drosophila melanogaster embryos. We have also tested the efficiency with which various short double-stranded DNA segments are assembled into plectonemic joints by RecA protein with this assay and found 5- to 10-fold differences. These results are interpreted as evidence for DNA sequence-specific effects in RecA-mediated homologous pairing in vitro.

Full text

PDF
5858

Images in this article

5856Image
on p.5856
5856Image
on p.5856
5856Image
on p.5856
5856Image
on p.5856
5856Image
on p.5856
5856Image
on p.5856
5857Image
on p.5857

Selected References

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

  1. Bianchi M., DasGupta C., Radding C. M. Synapsis and the formation of paranemic joints by E. coli RecA protein. Cell. 1983 Oct;34(3):931–939. doi: 10.1016/0092-8674(83)90550-0. [DOI] [PubMed] [Google Scholar]
  2. Cox M. M., Lehman I. R. Enzymes of general recombination. Annu Rev Biochem. 1987;56:229–262. doi: 10.1146/annurev.bi.56.070187.001305. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Cox M. M., Lehman I. R. recA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein. J Biol Chem. 1982 Jul 25;257(14):8523–8532. [PubMed] [Google Scholar]
  5. Cunningham R. P., Berger H. Mutations affecting genetic recombination in bacteriophage T4D. I. Pathway analysis. Virology. 1977 Jul 1;80(1):67–82. doi: 10.1016/0042-6822(77)90381-6. [DOI] [PubMed] [Google Scholar]
  6. Di Capua E., Müller B. The accessibility of DNA to dimethylsulfate in complexes with recA protein. EMBO J. 1987 Aug;6(8):2493–2498. doi: 10.1002/j.1460-2075.1987.tb02531.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunn K., Chrysogelos S., Griffith J. Electron microscopic visualization of recA-DNA filaments: evidence for a cyclic extension of duplex DNA. Cell. 1982 Apr;28(4):757–765. doi: 10.1016/0092-8674(82)90055-1. [DOI] [PubMed] [Google Scholar]
  8. Fishel R. A., Detmer K., Rich A. Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography. Proc Natl Acad Sci U S A. 1988 Jan;85(1):36–40. doi: 10.1073/pnas.85.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hotta Y., Tabata S., Bouchard R. A., Piñon R., Stern H. General recombination mechanisms in extracts of meiotic cells. Chromosoma. 1985;93(2):140–151. doi: 10.1007/BF00293161. [DOI] [PubMed] [Google Scholar]
  10. Hsieh P., Meyn M. S., Camerini-Otero R. D. Partial purification and characterization of a recombinase from human cells. Cell. 1986 Mar 28;44(6):885–894. doi: 10.1016/0092-8674(86)90011-5. [DOI] [PubMed] [Google Scholar]
  11. Julin D. A., Riddles P. W., Lehman I. R. On the mechanism of pairing of single- and double-stranded DNA molecules by the recA and single-stranded DNA-binding proteins of Escherichia coli. J Biol Chem. 1986 Jan 25;261(3):1025–1030. [PubMed] [Google Scholar]
  12. Keene M. A., Corces V., Lowenhaupt K., Elgin S. C. DNase I hypersensitive sites in Drosophila chromatin occur at the 5' ends of regions of transcription. Proc Natl Acad Sci U S A. 1981 Jan;78(1):143–146. doi: 10.1073/pnas.78.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kenne K., Ljungquist S. A DNA-recombinogenic activity in human cells. Nucleic Acids Res. 1984 Apr 11;12(7):3057–3068. doi: 10.1093/nar/12.7.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kmiec E. B., Holloman W. K. Heteroduplex formation and polarity during strand transfer promoted by Ustilago rec 1 protein. Cell. 1983 Jul;33(3):857–864. doi: 10.1016/0092-8674(83)90028-4. [DOI] [PubMed] [Google Scholar]
  15. Kmiec E., Holloman W. K. Homologous pairing of DNA molecules promoted by a protein from Ustilago. Cell. 1982 Jun;29(2):367–374. doi: 10.1016/0092-8674(82)90153-2. [DOI] [PubMed] [Google Scholar]
  16. Kolodner R., Evans D. H., Morrison P. T. 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. doi: 10.1073/pnas.84.16.5560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. McEntee K. Protein X is the product of the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5275–5279. doi: 10.1073/pnas.74.12.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McEntee K., Weinstock G. M., Lehman I. R. Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2615–2619. doi: 10.1073/pnas.76.6.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Muniyappa K., Shaner S. L., Tsang S. S., Radding C. M. Mechanism of the concerted action of recA protein and helix-destabilizing proteins in homologous recombination. Proc Natl Acad Sci U S A. 1984 May;81(9):2757–2761. doi: 10.1073/pnas.81.9.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shibata T., DasGupta C., Cunningham R. P., Radding C. M. Purified Escherichia coli recA protein catalyzes homologous pairing of superhelical DNA and single-stranded fragments. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1638–1642. doi: 10.1073/pnas.76.4.1638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wu A. M., Bianchi M., DasGupta C., Radding C. M. Unwinding associated with synapsis of DNA molecules by recA protein. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1256–1260. doi: 10.1073/pnas.80.5.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Yonesaki T., Minagawa T. T4 phage gene uvsX product catalyzes homologous DNA pairing. EMBO J. 1985 Dec 1;4(12):3321–3327. doi: 10.1002/j.1460-2075.1985.tb04083.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES