Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

• Published in: Methods (San Diego, Calif.) Calif.), 2012-06, Vol.57 (2), p.203-213
• Main Authors: Gillespie, Peter J., Gambus, Agnieszka, Blow, J. Julian
• Format: Article
• Language: English
• Subjects: Amphibia; Animals; Cell cycle; Cell Extracts - genetics; Cell Extracts - isolation & purification; Cell Nucleus - metabolism; Cell-free system; Chemical Precipitation; Chromatin; Chromatin - genetics; Chromatin - isolation & purification; Chromatin - metabolism; DNA - genetics; DNA - isolation & purification; DNA - metabolism; DNA biosynthesis; DNA Replication; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Egg extract; Eggs; Female; In vitro; Integration; Male; Mitosis; Nuclear Transfer Techniques; Nuclei; Oocytes - chemistry; Oocytes - cytology; Replication; sister chromatids; Sperm; Spermatozoa - cytology; Testis - cytology; Trichloroacetic Acid - chemistry; Xenopus; Xenopus laevis; Xenopus Proteins - isolation & purification; Xenopus Proteins - metabolism
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2012.03.029

The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.