Substrate specificity of the SpCCE1 holliday junction resolvase of Schizosaccharomyces pombe.
SpCCE1 from Schizosaccharomyces pombe is an endonuclease that resolves Holliday junctions in vitro. SpCCE1 also binds and cleaves a range of other DNAs (Y-junction; flap; and flayed, nicked, and partial duplexes) with varying efficiency. Cleavage sites are always 3' of thymine nucleotides positioned at or close to the branch point or strand interruption. SpCCE1's favored substrate is the X-junction. Up to two dimers of SpCCE1 can bind concurrently to the same X-junction at its crossover point. From mixing experiments of SpCCE1 and the Escherichia coli RuvA protein, we show that each dimer of SpCCE1 binds to a different face of the X-junction and that both are seemingly competent for strand cleavage. We propose that this provides a mechanism whereby SpCCE1 can scrutinize all four junction strands simultaneously for cleavable thymine nucleotides. SpCCE1 appears to resolve X-junctions by a nick and counter-nick mechanism. Therefore, to ensure a high probability of bilateral strand cleavage, SpCCE1 has a relatively long lifetime on X-junctions. This mechanism has the drawback of limiting dissociation from noncleavable junctions. We discuss why this might not be a problem in vivo.
Whitby
M C
MC
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. whitby@bioch.ox.ac.uk
Dixon
J
J
D001483:Base Sequence; D018983:DNA Footprinting; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D045565:Holliday Junction Resolvases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000201:enzymology*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases