Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (443K) 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 (11)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Woodfield, G.
Right arrow Articles by Burgin, A. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woodfield, G.
Right arrow Articles by Burgin, A. B.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2000, Vol. 28, No. 17 3323-3331
© 2000 Oxford University Press

Vaccinia topoisomerase and Cre recombinase catalyze direct ligation of activated DNA substrates containing a 3'-para-nitrophenyl phosphate ester

George Woodfield, Chonghui Cheng1, Stewart Shuman1 and Alex B. Burgin*

Biology Department, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4614, USA and 1Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA

DNA topoisomerases and DNA site-specific recombinases are involved in a diverse set of cellular processes but both function by making transient breaks in DNA. Type IB topoisomerases and tyrosine recombinases cleave DNA by transesterification of an active site tyrosine to generate a DNA–3'-phosphotyrosyl–enzyme adduct and a free 5'-hydroxyl (5'-OH). Strand ligation results when the 5'-OH attacks the covalent complex and displaces the enzyme. We describe the synthesis of 3'-phospho-(para-nitrophenyl) oligonucleotides (3'-pNP DNAs), which mimic the natural 3'-phosphotyrosyl intermediate, and demonstrate that such pre-activated strands are substrates for DNA ligation by vaccinia topoisomerase and Cre recombinase. Ligation occurs by direct attack of a 5'-OH strand on the 3'-pNP DNA (i.e., without a covalent protein–DNA intermediate) and generates free para-nitrophenol as a product. The chromogenic DNA substrate allows ligation to be studied in real-time and in the absence of competing cleavage reactions and can be exploited for high-throughput screening of topoisomerase/recombinase inhibitors.

* To whom correspondence should be addressed. Tel: +1 619 594 0533; Fax: +1 619 594 5676; Email: aburgin@sunstroke.sdsu.edu


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
 Nucleic Acids ResHome page
K. Malanowska, S. Yoneji, A. A. Salyers, and J. F. Gardner
CTnDOT integrase performs ordered homology-dependent and homology-independent strand exchanges
Nucleic Acids Res., September 27, 2007; 35(17): 5861 - 5873.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
S. Antony, C. Marchand, A. G. Stephen, L. Thibaut, K. K. Agama, R. J. Fisher, and Y. Pommier
Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1
Nucleic Acids Res., July 26, 2007; 35(13): 4474 - 4484.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. C. Rideout, A. C. Raymond, and A. B. Burgin Jr
Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I
Nucleic Acids Res., August 27, 2004; 32(15): 4657 - 4664.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
R. A. Kazmierczak, B. M. Swalla, A. B. Burgin, R. I. Gumport, and J. F. Gardner
Regulation of site-specific recombination by the C-terminus of {lambda} integrase
Nucleic Acids Res., December 1, 2002; 30(23): 5193 - 5204.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. D. Bromberg, C. Hendricks, A. B. Burgin, and N. Osheroff
Human Topoisomerase IIalpha Possesses an Intrinsic Nucleic Acid Specificity for DNA Ligation. USE OF 5' COVALENTLY ACTIVATED OLIGONUCLEOTIDE SUBSTRATES TO STUDY ENZYME MECHANISM
J. Biol. Chem., August 16, 2002; 277(34): 31201 - 31206.
[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.