Skip Navigation

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by WATABE, H.-o.
Right arrow Articles by ANDO, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by WATABE, H.-o.
Right arrow Articles by ANDO, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

J. Biochem, 1984, Vol. 95, No. 6 1677-1690
© 1984 Japanese Biochemical Society

research-article

Purification of a Eukaryotic Site-Specific Endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and Effectors on Its Specificity and Activity1

Hiro-omi WATABE, Takehiko SHIBATA2, Tohru IINO3 and Tadahiko ANDO

Department of Microbiology, Riken Institute (Institute of Physical and Chemical Research) Wako, Saitama 351

3To whom correspondence should be addressed

A site-specific endonuclease (Endo.Sce 1) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double- stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.

1This work was partly supported by grants for "Life Science" and for "Promotion of Science and Technology" from the Science and Technology Agency of Japan, and partly by a grant from the Ministry of Education, Science and Culture of Japan.

2Present address: Pharmaceutical Research Laboratories, Meiji Seika, Ltd., Kohoku-ku, Yokohama, Kanagawa 222.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.