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Synthetic junctions as tools to identify and characterize Holliday junction resolvases
Genetic exchanges between chromosomes can lead to the formation of DNA intermediates known as Holliday junctions. The structure of these intermediates has been determined both biochemically and structurally, and their interactions with Holliday junction processing enzymes have been well characterized. A number of proteins, from both prokaryotic and eukaryotic sources, have been identified that promote the nucleolytic resolution of junctions. To facilitate these studies, synthetic DNA substrates that mimic true Holliday junctions have been developed. These now provide an important resource for both the identification and the characterization of novel Holliday junction resolvase activities. This chapter describes methods detailing the preparation and use of synthetic Holliday junctions and how they are best utilized in the study of proteins that might exhibit resolvase activity. Additionally, a method is described that can be used to rapidly screen a TAP-tagged library of proteins for resolvase activity without a need for conventional purification procedures.
History
Publication status
- Published
Journal
Methods in EnzymologyISSN
0076-6879Publisher
ElsevierExternal DOI
Volume
408Page range
485-501Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Research groups affiliated with
- Genome Damage and Stability Centre Publications
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- No
Peer reviewed?
- Yes
Legacy Posted Date
2018-09-26Usage metrics
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