Holliday junctions (four-way DNA junctions), formed during homologous recombination, are bound and resolved by junction-specific endonucleases to yield recombinant duplex DNA products. The junction-resolving enzymes are a structurally diverse class of proteins that nevertheless have many properties in common; in particular a high structure specificity for binding and metal-dependent, (frequently) sequence-specific cleavage activity. In Saccharomyces cerevisiae, the enzyme CCE1 is necessary for the resolution of recombining mitochondrial genomes, and in Schizosaccharomyces pombe the homologous protein YDC2 is thought to have a similar function. We have generated an inactive mutant of YDC2, D226N, that retains structure-specific junction binding and have analysed the interaction of this protein with the four-way DNA junction. YDC2 binds the four-way junction in two specific complexes (I and II), unfolding the stacked X-structure into a conformation where the arms extend to the four corners of a square. This structure is reminiscent of that of the free junction in the absence of metal ions and of the structures imposed on the Holliday junction by CCE1 and RuvA. DNase I probing reveals footprints specific for complexes I and II which extend from the junction centre on all four arms. No protection is observed with the small, hydrophobic probe DMS
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