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.
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.
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004277:DNA, Single-Stranded; D004247:DNA; D011938:Rec A Recombinases; D011088:DNA Ligases