利用纯化的芽殖酵母蛋白进行真核生物DNA复制
Eukaryotic DNA replication with purified budding yeast proteins.
作者信息
Posse Viktor, Johansson Erik, Diffley John F X
机构信息
Chromosome Replication Laboratory, The Francis Crick Institute, London, United Kingdom; Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
出版信息
Methods Enzymol. 2021;661:1-33. doi: 10.1016/bs.mie.2021.08.018. Epub 2021 Oct 22.
The in vitro reconstitution of origin firing was a key step toward the biochemical reconstitution of eukaryotic DNA replication in budding yeast. Today the basic replication assay involves proteins purified from 24 separate protocols that have evolved since their first publication, and as a result, the efficiency and reliability of the in vitro replication system has improved. Here we will present protocols for all 24 purifications together with a general protocol for the in vitro replication assay and some tips for troubleshooting problems with the assay.
体外复制起点激活的重建是在芽殖酵母中对真核生物DNA复制进行生化重建的关键一步。如今，基本的复制测定涉及从24个不同方案中纯化的蛋白质，这些方案自首次发表以来不断演变，因此，体外复制系统的效率和可靠性得到了提高。在这里，我们将介绍所有24种纯化方法的方案，以及体外复制测定的通用方案，还有一些针对该测定问题的故障排除提示。
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