Yeast resolving enzyme CCE1 makes sequential cleavages in DNA junctions within the lifetime of the complex.
CCE1 is a DNA junction-resolving enzyme of Saccharomyces cerevisiae. Such enzymes are required to make two symmetrically paired cleavages in order to resolve the four-way junction productively. Using a cruciform assay, we show here that CCE1 introduces two unilateral cleavages in a sequential manner. This requires that the protein remains bound to the junction, preventing branch migration of the point of strand exchange. From a detailed kinetic analysis, we find that the CCE1 cleavage at a given site is accelerated by a factor of 5-10 when it occurs subsequently to the initial cleavage. These properties ensure a productive resolution of the four-way junction and may be general for junction-resolving enzymes.
Fogg
J M
JM
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, U.K.
Schofield
M J
MJ
Déclais
A C
AC
Lilley
D M
DM
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045565:Holliday Junction Resolvases; D007700:Kinetics; D011485:Protein Binding; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases