Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
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 ISI Web of Science
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 Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Haruki, M.
Right arrow Articles by Kanaya, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Haruki, M.
Right arrow Articles by Kanaya, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 13, No. 12, 881-886, December 2000
© 2000 Oxford University Press

Efficient cleavage of RNA at high temperatures by a thermostable DNA-linked ribonuclease H

Mitsuru Haruki, Tomoko Nogawa, Nobutaka Hirano, Hyongi Chon, Yasuo Tsunaka, Masaaki Morikawa and Shigenori Kanaya,1

Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2–1, Yamadaoka, Suita, Osaka 565-0871, Japan

To construct a DNA-linked RNase H, which cleaves RNA site-specifically at high temperatures, the 15-mer DNA, which is complementary to the polypurine-tract sequence of human immunodeficiency virus-1 RNA (PPT-RNA), was cross-linked to the unique thiol group of Cys135 in the Thermus thermophilus RNase HI variant. The resultant DNA-linked enzyme (d15-C135/TRNH), as well as the d15-C135/ERNH, in which the RNase H portion of the d15-C135/TRNH is replaced by the Escherichia coli RNase HI variant, cleaved the 15-mer PPT-RNA site-specifically. The mixture of the unmodified enzyme and the unlinked 15-mer DNA also cleaved the PPT-RNA but in a less strict manner. In addition, this mixture cleaved the PPT-RNA much less effectively than the DNA-linked enzyme. These results indicate that the cross-linking limits but accelerates the interaction between the enzyme and the DNA/RNA substrate. The d15-C135/TRNH cleaved the PPT-RNA more effectively than the d15-C135/ERNH at temperatures higher than 50°C. The d15-C135/TRNH showed the highest activity at 65°C, at which the d15-C135/ERNH showed little activity. Such a thermostable DNA-linked RNase H may be useful to cleave RNA molecules with highly ordered structures in a sequence-specific manner.


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


This article has been cited by other articles:


Home page
 Protein Eng Des SelHome page
H. Chon, Y. Tsunaka, M. Haruki, M. Morikawa, and S. Kanaya
Site-specific cleavage of MS2 RNA by a thermostable DNA-linked RNase H
Protein Eng. Des. Sel., August 1, 2002; 15(8): 683 - 688.
[Abstract] [Full Text] [PDF]


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.