Homologous recombination intermediates between two duplex DNA catalysed by human cell extracts

Open Access
Publisher Website
Google Scholar
Abstract
Using as substrates, 1: the replicative form (RF) of phage M13 mp8 in which the reading frame of the lacZ′ gene was disrupted by insertion of an octonucleotide, and 2: a restriction fragment one kb long, containing the functional lacZ′ gene (isolated from wild type M13 mp8), we show that nuclear extracts from human cells (3 lines tested) promote the targeted replacement of the altered sequence by the functional one. Following incubation with the extracts, the DNA's were introduced in JM 109 bacteria (rec A− and lac Z′−) which were grown in presence of a colorimetric indicator of β-galactosidase activity. Homologous recombination gives rise to the genotypical modification : lacZ′+ instead of lacZ′ in the bacteriophage DNA. This is revealed by phenotypical expression of the lacZ′ gene product in replicating bacteriophage, i. e. the formation of blue instead of white plaques. The frequency of recombination (blue/total plaques) is increased by a factor of 50–80 as a function of protein concentration and of incubation time. The maximal frequency observed is 5×10−. There is no increase over the background when extracts are boiled. Electrophoresis and electron microscopy of DNA's incubated with the extracts show the formation of recombination intermediates with single strand exchange. Restriction analysis of recombined DNA confirms that the process corresponds to targeted sequence exchange. These data allow to propose three steps for homologous recombination between two duplex DNA's : i) unpairing of the two duplexes; ii) single-strand exchange and synaptic pairing; iii) resolution of the cross-junctions. The three steps correspond to those predicted by the gene conversion model of Holliday.