Cell Cycle Synchronization in Xenopus Egg Extracts

Abstract

Many important discoveries in cell cycle research have been made using cell-free extracts prepared from the eggs of the South African clawed frog Xenopus laevis. These extracts efficiently support the key nuclear functions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. 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. Here, we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei. We detail how these extracts can be used to study the key transitions of the eukaryotic cell cycle and describe conditions under which these transitions can be manipulated by addition of drugs that either retard or advance passage. In addition, we describe in detail essential techniques that provide a practical starting point for investigating the function of proteins involved in the operation of the eukaryotic cell cycle.

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

“…Metaphase-arrested Xenopus laevis egg extract and demembranated Xenopus sperm nuclei were prepared and used as described previously (Gillespie et al., 2016). Briefly, extracts were released into interphase from metaphase arrest once incubated with 0.3 mM CaCl 2 for 15 min at 23°C.…”

“…For DNA synthesis reactions, sperm nuclei were incubated at 3–10 ng DNA/μL in extract. DNA synthesis was assayed by measuring incorporation of [α- 32 P]dATP into acid-insoluble material followed by scintillation counting (Gillespie et al., 2016). …”

Xenopus egg extract to study regulation of genome‐wide and locus‐specific DNA replication

“…The first will give rise to a mitotic extract (or CSF‐arrested extract) that can support spindle assembly, chromosome condensation and segregation, which can be driven into interphase by addition of CaCl 2 . The second will lead to an interphase extract, that supports chromatin decondensation, nuclear assembly and semiconservative DNA replication (Gillespie, Neusiedler, Creavin, Chadha, & Blow, ). Further ultracentrifugation of low‐speed extracts (LSS) will result in high‐speed supernatant (HSS) of egg extracts, that are able to assemble chromatin on duplex DNA (Almouzni & Mechali, ) and support replication of single‐stranded DNA (ssDNA) plasmid substrates.…”

“…Xenopus egg extracts are particularly suitable to dissect the different steps of DNA replication, due to the availability of standardized treatments and inhibitors (Gillespie et al, 2016) and to the absence of transcription. In the past decades, this system gave a major contribution to the isolation and biochemical characterization of proteins and molecular complexes involved in DNA replication, such as CDK and DDK (Fang & Newport, 1991), MCM helicase (Chong, Mahbubani, Khoo, & Blow, 1995), the licensing inhibitor Geminin (McGarry & Kirschner, 1998) and other replisome components.…”

Xenopus cell-free extracts and their contribution to the study of DNA replication and other complex biological processes

“…For example, mass spectrometry provides a powerful way to identify proteins in large ultrastructures, such as nuclear pores, replisomes, and chromosomes that can be assembled in vitro and then carefully separated for analysis of their contents (Gambus et al, 2011;Gillespie et al, 2012). There are now a large number of procedures that can be used to create and control cell cycle synchrony in egg extracts with unprecedented precision (Gillespie et al, 2016). Xenopus egg extracts can be used for live microscopy imaging, and in future this approach could be developed to deliver large amounts of data and 'high content' analysis of processes that can be followed by fluorescently marked components.…”