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.
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.
- Publication:
-
Proceedings of the National Academy of Science
- Pub Date:
- August 1988
- DOI:
- 10.1073/pnas.85.16.5854
- Bibcode:
- 1988PNAS...85.5854M