In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.
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 MAT alpha 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.
Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, California 92307.
J A
JA
Nickoloff
J D
JD
Singer
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F
Heffron
D001483:Base Sequence; D003196:Computer Graphics; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000648:ultrastructure; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004271:DNA, Fungal; D004274:DNA, Recombinant; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific