The role of the SAP motif in promoting Holliday junction binding and resolution by SpCCE1.
Holliday junctions are four-way branched DNA structures that are formed during recombination and by replication fork regression. Their processing depends on helicases that catalyze junction branch migration, and endonucleases that resolve the junction into nicked linear DNAs. Here we have investigated the role of a DNA binding motif called SAP in binding and resolving Holliday junctions by the fission yeast mitochondrial resolvase SpCCE1. Mutation or partial/complete deletion of the SAP motif dramatically impairs the ability of SpCCE1 to resolve Holliday junctions in a heterologous in vivo system. These mutant proteins retain the ability to recognize the junction structure and to distort it upon binding. However, once formed the mutant protein-junction complexes are relatively unstable and dissociate much faster than wild-type complexes. We show that binding stability is necessary for efficient junction resolution, and that this may be due in part to a requirement for maintaining the junction in an open conformation so that it can branch migrate to cleavable sites.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Jong Sook
JS
Ahn
Matthew C
MC
Whitby
D000595:Amino Acid Sequence; D001665:Binding Sites; D003460:Crystallization; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000378:metabolism; D004355:Drug Stability; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D045565:Holliday Junction Resolvases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins; D012568:Schizosaccharomyces / Q000201:enzymology; D013329:Structure-Activity Relationship
D004272:DNA, Mitochondrial; D011994:Recombinant Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases