Targeting a truncated Ho-endonuclease of yeast to novel DNA sites with foreign zinc fingers.

AUTOR(ES)

Nahon, E

RESUMO

Ho-endonuclease of the yeast, Saccharomyces cerevisiae, initiates a mating type switch by making a site-specific double strand break in the mating type gene, MAT. Ho is a dodecamer endonuclease and shares six of the seven intein motifs with PI- Sce I endonuclease, an intein encoded by the VMAI gene. We show that a 113 residue truncated Ho-endonuclease starting at intein motif C initiates a mating type switch in yeast. Ho is the only dodecamer endonuclease with zinc fingers. To see whether they have a role in determining site specificity we exchanged them for zinc fingers of the yeast transcription factor, Swi5. A chimeric endonuclease comprising the dodecamer motifs of Ho (C-E) and the zinc finger domain of Swi5 cleaves a Swi5 substrate plasmid in vivo. A similar chimera with the zinc fingers of SpI cleaves a GC box rich substrate plasmid. These experiments delineate a catalytic fragment of Ho-endonuclease that can be fused to various DNA binding moieties in the design of chimeric endonucleases with new site specificities.

Documentos Relacionados

• Basonuclin: a keratinocyte protein with multiple paired zinc fingers.
• Stereochemical basis of DNA recognition by Zn fingers.
• The KUP gene, located on human chromosome 14, encodes a protein with two distant zinc fingers.
• glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers.
• A human alpha-fetoprotein enhancer-binding protein, ATBF1, contains four homeodomains and seventeen zinc fingers.