Interaction of the resolving enzyme YDC2 with the four-way DNA junction.
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.
White
M F
MF
Department of Biochemistry, University of Dundee, Dundee DD1 5EH, UK. mfwhite@bad.dundee.ac.uk
Lilley
D M
DM
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins
D029702:Schizosaccharomyces pombe Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C108766:cce1 protein, S pombe