Abstract:
:Oligonucleotide-directed triple helix formation provides an elegant rational basis for gene-specific DNA targeting and has been widely used to interfere with gene expression ("antigene" strategies) and as a molecular tool for biological studies. Various strategies have been developed to introduce sequence modifications in genomes. However, the low efficiency of the overall process in eucaryotic cells impairs efficient recovery of recombinant genomes. Since one limiting step in homologous recombination is the targeting to the homologous sequence, we have tested the contribution of an oligonucleotide-directed triple helix formation on the RecA-dependent association of an oligonucleotide and its homologous target on duplex DNA (D-loop formation). For this study, the recombinant ssDNA fragment was noncovalently associated to a triple helix-forming oligonucleotide. The physicochemical and biochemical characteristics of the triple helix and D-loop structures formed by the complex molecules in the presence or in the absence of RecA protein were determined. We have demonstrated that the triple helix-forming oligonucleotide increases the efficiency of D-loop formation and the RecA protein speeds up also the triple helix formation. The so-called "GOREC" (for guided homologous recombination) approach can be developed as a novel tool to improve the efficiency of directed mutagenesis and gene alteration in living organisms.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Biet E,Maurisse R,Dutreix M,Sun Jsdoi
10.1021/bi001605asubject
Has Abstractpub_date
2001-02-13 00:00:00pages
1779-86issue
6eissn
0006-2960issn
1520-4995pii
bi001605ajournal_volume
40pub_type
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