Abstract
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.
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Bachrati, C.Z., Hickson, I.D. (2009). Dissolution of Double Holliday Junctions by the Concerted Action of BLM and Topoisomerase IIIα. In: Clarke, D. (eds) DNA Topoisomerases. Methods in Molecular Biology™, vol 582. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-340-4_8
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DOI: https://doi.org/10.1007/978-1-60761-340-4_8
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