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M602933200v1
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Papers In Press, published online ahead of print April 11, 2006
J. Biol. Chem, 10.1074/jbc.M602933200
Submitted on March 28, 2006
Accepted on April 10, 2006

A novel, topologically constrained DNA molecule containing a double Holliday junction: Design, synthesis and initial biochemical characterization

Jody L. Plank and Tao-shih Hsieh

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710-0001

Corresponding Author: hsieh{at}biochem.duke.edu

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 double Holliday junction substrate (DHJS) that consists of two small, double-stranded DNA circles conjoined by two Holliday junctions (HJ’s). Its biochemical synthesis is based on the production of 2 pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJ’s is identical on both strands, allowing the HJ’s to migrate without the generation of unpaired regions of DNA, while the distance between the HJ’s 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 DHJS 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.


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