HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 14, 12089-12098, April 5, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/14/12089    most recent M111315200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bao, K. K. Articles by Wong, I. Search for Related Content PubMed PubMed Citation Articles by Bao, K. K. Articles by Wong, I. Social Bookmarking                      What's this?

Presteady-state Analysis of Avian Sarcoma Virus Integrase
I. A SPLICING ACTIVITY AND STRUCTURE-FUNCTION IMPLICATIONS FOR COGNATE SITE RECOGNITION^*

Kogan K. Bao, Anna Marie Skalka^§¶, and Isaac Wong

From the  Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331 and ^§ Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends. This splicing reaction showed an initial exponential phase (k_splicing = 0.02 s¹) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred. With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products. Furthermore, the splicing reaction allowed characterization of substrate binding and specificity. Although integrase requires only a 3' hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile. These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				I. Wong, A. S. Bernards, J. K. Miller, and J. A. Wirz A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase J. Biol. Chem., January 17, 2003; 278(4): 2411 - 2418. [Abstract] [Full Text] [PDF]

				K. K. Bao, H. Wang, J. K. Miller, D. A. Erie, A. M. Skalka, and I. Wong Functional Oligomeric State of Avian Sarcoma Virus Integrase J. Biol. Chem., January 3, 2003; 278(2): 1323 - 1327. [Abstract] [Full Text] [PDF]

				A. S. Bernards, J. K. Miller, K. K. Bao, and I. Wong Flipping Duplex DNA Inside Out. A DOUBLE BASE-FLIPPING REACTION MECHANISM BY ESCHERICHIA COLI MutY ADENINE GLYCOSYLASE J. Biol. Chem., May 31, 2002; 277(23): 20960 - 20964. [Abstract] [Full Text] [PDF]

				K. K. Bao, A. M. Skalka, and I. Wong Presteady-state Analysis of Avian Sarcoma Virus Integrase. II. REVERSE-POLARITY SUBSTRATES IDENTIFY PREFERENTIAL PROCESSING OF THE U3-U5 PAIR J. Biol. Chem., March 29, 2002; 277(14): 12099 - 12108. [Abstract] [Full Text] [PDF]