Authors
Dmitry V Bugreev, Olga M Mazina, and Alexander V Mazin
Summary
The Holliday junction is a key intermediate in various genetic processes including homologous and site-specific recombination and DNA replication1-3. Holliday junction is able to branch migrate along DNA allowing the exchange between homologous DNA regions. In bacteria, there are enzymes that promote DNA branch migration (BM)3. In eukaryotes, we recently showed that Rad54 protein catalyses BM of Holliday junctions4. Previously, “non-movable” X-junctions, consisting of a central homologous “movable” core flanked by the mutually heterologous terminal DNA branches5 (Fig. 1a), were successfully used for detection of the BM activities of prokaryotic enzymes (ruvAB, RecG) that have both BM and helicase activities3. However they became unsuitable for detection of BM activity of Rad54 that do not have helicase activity4. We therefore constructed a “movable” X-junction in which three of the four terminal DNA branches were mutually homologous, allowing the crossover point to move freely by BM up to complete separation of two DNA duplexes, without any need for a helicase activity (Fig. 1b). Here we describe the protocols for construction of branched DNA substrates including the X-junctions (Holliday junctions), PX-junctions (partial junctions), movable replication forks and for usage of these substrates for detection of both 4-stranded and 3-standed BM activities of proteins.Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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