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J. Biol. Chem., Vol. 281, Issue 25, 17510-17516, June 23, 2006

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A Novel, Topologically Constrained DNA Molecule Containing a Double Holliday Junction

DESIGN, SYNTHESIS, AND INITIAL BIOCHEMICAL CHARACTERIZATION^*

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

The double Holliday junction (dHJ) is a central intermediate to homologous recombination, but biochemical analysis of the metabolism of this structure has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a novel dHJ substrate that consists of two small, double stranded DNA circles conjoined by two Holliday junctions (HJs). Its biochemical synthesis is based on the production of two pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJs is identical on both strands, allowing the HJs to migrate without the generation of unpaired regions of DNA, whereas the distance between the HJs is on the order of gene conversion tracts thus far measured in Drosophila and mouse model systems. The structure of this substrate also provides similar topological constraint as would occur in an endogenous dHJ. Digestion of the dHJ substrate by T7 endonuclease I resolves the substrate into crossover and non-crossover products, as predicted by the Szostak model of double strand break repair. This substrate will greatly facilitate the examination of the mechanism of resolution of double Holliday junctions.

Received for publication, March 28, 2006

^* This work was supported by Grant GM29006 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed. Tel.: 919-684-6501; E-mail: hsieh{at}biochem.duke.edu.

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				M. R. Webb, J. L. Plank, D. T. Long, T.-s. Hsieh, and K. N. Kreuzer The Phage T4 Protein UvsW Drives Holliday Junction Branch Migration J. Biol. Chem., November 23, 2007; 282(47): 34401 - 34411. [Abstract] [Full Text] [PDF]

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