In the past decade, site-specific chromosomal DNA cleavage mediated by DNA endonucleases has been used to examine diverse aspects of chromosome structure and function in eukaryotes, such as DNA topology, replication, transcription, recombination, and repair. Here we describe a method with which chromosomes can be linearized at any predefined position in vivo. Yeast homothallic switching endonuclease (HO endo), a sequence-specific double-strand nuclease involved in mating-type switching, is employed for targeting DNA cleavage. HO endo contains discrete functional domains: a N-terminal nuclease and a C-terminal DNA-binding domain, thereby allowing construction of a chimeric nuclease with the cutting site distinct from the original HO recognition sequence. The expression of the nuclease is engineered to be controlled by a tightly regulated, inducible promoter. The cut sites recognized by HO endo or its derivatives are introduced specifically at desired positions in the yeast genome by homologous recombination. Here we present experimental procedures and review some applications based on this approach in yeast and other biological systems.
Copyright 1999 Academic Press.