Homology-dependent cutting in trans of DNA in extracts of Escherichia coli: an approach to the enzymology of genetic recombination

Abstract

An in vitro system is described in which the cutting of crosslinked phiX replicative form (RF) I DNA molecules by the uvr system of Escherichia coli induces the cutting of homologous undamaged DNA during incubation with crude extracts of thermally induced E. coli (lambda precA+) lysogens. This reaction, which has also been observed in intact E. coli lysogens infected with lambda phages, is dependent on the presence of functional recA+ and uvrB+ gene products. Extracts from thermally induced lambda precA+ lysogens of E. coli proved to be substantially more active than extracts from nonlysogenic cells of the same strain. The results provide preliminary evidence for an endonuclease activity that cuts intact superhelical DNA in response to interaction with homologus damaged DNA. In the present paper, we describe an in vitro system in which both the endonucleolytic cutting of DNA containing crosslinks and the induced cutting of undamaged DNA can be studied without purification of the participating enzymes. Although the information obtained is fragmentary and often puzzling, we feel that this system can contribute to an understanding of the complex mechanisms involved in repair and recombination.