Dissolution of double Holliday junctions by the concerted action of BLM and topoisomerase IIIalpha.
In eukaryotic cells, topoisomerase III forms an evolutionarily conserved complex with a RecQ family helicase and two OB-fold containing proteins, replication protein A (RPA) and RMI1. One role for this complex is to catalyze the completion of homologous recombination reactions in which the recombining DNA molecules are covalently interlinked by a double Holliday junction structure. This process, which requires the single-stranded DNA decatenation activity of topoisomerase III, is termed Holliday junction "dissolution" to distinguish it from Holliday junction "resolution" catalyzed by endonucleases (resolvases) that simply cleave the four-way junction. Holliday junction dissolution gives rise exclusively to non-cross-over recombinant products, which would have the effect of suppressing sister chromatid exchanges and loss of heterozygosity between homologous chromosomes. In this chapter, we provide a detailed experimental protocol for the preparation of an oligonucleotide-based, double Holliday junction substrate and for the biochemical analysis of dissolution in vitro.
Bachrati
Csanád Z
CZ
Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.
Hickson
Ian D
ID
D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D045566:DNA, Cruciform / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism*
D002352:Carrier Proteins; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; C501527:RMI1 protein, human; C097151:Bloom syndrome protein; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I