Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence (original) (raw)

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1

Department of Biology

Baltimore, MD 21218, USA

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2

Department of Chemistry, The Johns Hopikins Univesity

Baltimore, MD 21218, USA

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,

3

Department of Microbiology, University of Texas

Austin, TX 78712, USA

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1

Department of Biology

Baltimore, MD 21218, USA

2

Department of Chemistry, The Johns Hopikins Univesity

Baltimore, MD 21218, USA

*To Whom correspondence should be addressed

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+Present address: Department of Biochemistry, School of Public Health, The John Hopkins University, Baltimore, MD 21205, USA

§Present address: Department of Agronomy and Range Science, University of Califormia, Davis, CA 95616, USA

Author Notes

Published:

11 August 1995

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Amy S. Kimball, Melissa L. Kimball, Makkuni Jayaram, Thomas D. Tullius, Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence, Nucleic Acids Research, Volume 23, Issue 15, 11 August 1995, Pages 3009–3017, https://doi.org/10.1093/nar/23.15.3009
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Abstract

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly Identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationallycompetent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthrollne*copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of loweroccupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.

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Author notes

+Present address: Department of Biochemistry, School of Public Health, The John Hopkins University, Baltimore, MD 21205, USA

§Present address: Department of Agronomy and Range Science, University of Califormia, Davis, CA 95616, USA

© 1995 Oxford University Press

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