DNA segments encoding a domain of HO-endonuclease

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase

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536 232, 536 234, 435 697, C12N 922, C12N 1555, C12N 1562

Patent

active

060371625

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to DNA segments encoding the two functional domains of HO-endonuclease, the catalytic and DNA recognition domains.


BACKGROUND ART

HO-endonuclease belongs to the dodecapeptide family of endonucleases that cleave a large, unique (>18 bp) DNA recognition sequence that lacks dyad symmetry. These nucleases are encoded by nuclear or mitochondrial and chloroplast group I intron genes, or exist as inteins having their coding sequences embedded in-frame within unrelated genes that undergo protein splicing, such that two proteins are generated from a single translation product. Dodecapeptide endonucleases are known in phylogenetically diverse species, and new genes are constantly being discovered.
HO-endonuclease of the yeast Saccaromyces cerevisiae initiates a mating type switch by making a site-specific double strand break in the mating type gene MAT, and attains site-specificity by virtue of a large (18-24 bp) target site. It has now been shown that a 113-residue N-terminal truncation of HO-endonuclease is able to cleave its cognate site and to initiate a mating type switch in yeast.
HO is the only dodecapeptide endonuclease with a zinc finger DNA-binding domain. The present inventors have cloned an inactive allele of HO-endonuclease from a laboratory strain of yeast [Meiron, et al., "Identification of the Heterothallic Mutation in HO-endonuclease of S. cerevisiae Using HO/ho Chimeric Genes," Curr. Genet., Vol. 28, pp. 367-373 (1995)]. In that study, chimeric genes were made between the active and inactive endonuclease genes and tested for their activity in yeast. It was found that of the four amino acid substitutions in the mutant (ho) gene, only one substitution affected endonucleolytic activity. This mutation is in the first zinc finger of HO.
In the present invention, it is shown that it is possible to target endonucleolytic activity to a different site by exchange of the zinc finger domain of HO for that of the yeast transcription factor Swi5. It was found that a chimeric endonuclease comprising the nuclease domain of HO and the zinc finger domain of Swi5 cleaves the Swi5 target site URS1. This shows that it is possible to target the catalytic domain of dodecapeptide endonucleases via various DNA-binding moieties, and that they present a general nuclease domain in the design of highly site-specific targeted endonucleases.


SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide isolated domains of dodecapeptide endonucleases, in particular of HO-endonuclease, for chimeric nucleases that will be used as rare cutters in vitro for mapping and sequencing and in vivo for gene therapy and against pathogenic agents.
The present invention achieves the above objective by providing an isolated DNA segment encoding a domain of HO-endonuclease, said segment being selected from the group consisting of (a) an isolated DNA segment encoding the N-terminus of HO-endonuclease which contains the general catalytic nuclease activity of said HO-endonuclease, said DNA segment comprising at least 755 nt and encoding for at least 251 amino acids, and said endonuclease being characterized by the presence of at least one copy of the dodecapeptide motif LAGLI-DAIG; (SEQ ID NO:1) and (b) an isolated DNA segment encoding the C-terminus of HO-endonuclease which contains the DNA binding/recognition activity of said HO-endonuclease, said C-terminus comprising about 360 nt and encoding for about 120 amino acids.
In another aspect of the invention, there is provided a DNA construct comprising a DNA segment encoding the catalytic nuclease domain of a dodecapeptide endonuclease, a second DNA segment encoding a DNA binding moiety and a vector, said endonuclease being characterized by the presence of at least one copy of the dodecapeptide motif LAGLI-DADG (SEQ ID No:1)
In another aspect of the invention, there is provided a chimeric restriction endonuclease, comprising the catalytic nuclease domain of a dodecapeptide endonuclease linked to a recognitio

REFERENCES:
Nahon, E., et al. (1998) Nucl. Acid Res. 26(5), 1233-1239.
Meiron, H., et al. (1995) Curr. Genet. 28, 367-373.
Dohrmann, P.R., et al. (1992) Genes and Dev. 6(1), 93-104.
Gimble, F. S., et al. (1995) J. Biol. Chem. 270(11), 5849-5856.

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