In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis

Jac A. Nickoloff, Jeffrey D. Singer, Fred Heffron

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MATα or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.

Original languageEnglish (US)
Pages (from-to)1174-1179
Number of pages6
JournalMolecular and Cellular Biology
Volume10
Issue number3
StatePublished - 1990
Externally publishedYes

Fingerprint

Site-Directed Mutagenesis
Saccharomyces cerevisiae
Genetic Vectors
Mutation
Base Pairing
Genetic Recombination
wye

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. / Nickoloff, Jac A.; Singer, Jeffrey D.; Heffron, Fred.

In: Molecular and Cellular Biology, Vol. 10, No. 3, 1990, p. 1174-1179.

Research output: Contribution to journalArticle

Nickoloff, Jac A. ; Singer, Jeffrey D. ; Heffron, Fred. / In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis. In: Molecular and Cellular Biology. 1990 ; Vol. 10, No. 3. pp. 1174-1179.
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