SpringerProtocols: Abstract: Dissolution of Double Holliday Junctions by the Concerted Action of BLM and Topoisomerase III?

8. Dissolution of Double Holliday Junctions by the Concerted Action of BLM and Topoisomerase IIIα

By: Csanád Z. Bachrati¹, Ian D. Hickson¹

Abstract

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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.

Affiliation(s): (1) Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Book Title: DNA Topoisomerases: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 582 | Pub. Date: Sep-22-2009 | Page Range: 91-102 | DOI: 10.1007/978-1-60761-340-4_8

Subject: Genetics/Genomics

Key Words: Double Holliday junction dissolution - Bloom’s syndrome helicase - topoisomerase IIIα

References

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