DISSECTION OF THE SEQUENCE SPECIFICITY OF THE HOLLIDAY JUNCTION ENDONUCLEASE CCE1

CCEI is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomyc es cerevisiae. The junction-resolving enzymes are a diverse class, wid ely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single turnover ki netic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT down arrow A has been identified, with specificity residing mainly at the central CT d inucleotide. Equilibrium constants for CCE1 binding to four-way juncti ons are unaffected by sequence variations, suggesting that substrate d iscrimination occurs predominantly in the transition state complex. CC E1 cuts most efficiently at the junction center, but can also cleave t he DNA backbone at positions one nucleotide 3' or 5' of the point of s trand exchange, suggesting a significant degree of conformational flex ibility in the CCE1:junction complex. Introduction of base analogues a t single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 mo iety of the guanine base-pairing with the cytosine of the consensus se quence appears to be crucial for catalysis. The functional significanc e of sequence specificity in junction-resolving enzymes is discussed.