Abstract
Cell-free extracts from Xenopus laevis eggs are a model system for studying chromosome biology. Xenopus egg extracts can be synchronised in different cell cycle stages, making them useful for studying DNA replication, DNA repair and chromosome organisation. Combining single-molecule approaches with egg extracts is an exciting development being used to reveal molecular mechanisms that are difficult to study using conventional approaches. Fluorescence-based single-molecule imaging of surface-tethered DNAs has been used to visualise labelled protein movements on stretched DNA, the dynamics of DNA–protein complexes and extract-dependent structural rearrangement of stained DNA. Force-based single-molecule techniques are an alternative approach to measure mechanics of DNA and proteins. In this essay, the details of these single-molecule techniques, and the insights into chromosome biology they provide, will be discussed.
Highlights
For cells to remain healthy and properly divide, genome stability and appropriate gene expression must be maintained
Xenopus egg extracts are a powerful in vitro system as large volumes of cell-free extract synchronised in the cell cycle can reconstitute complex reactions involving chromosomes
Genetic engineering is not routine, extracts are manipulated through immunodepletion, chemical inhibition and addition of modified substrates
Summary
For cells to remain healthy and properly divide, genome stability and appropriate gene expression must be maintained. Alongside DNA packaging by histones, other proteins provide higher order chromosome organisation, notably the structural maintenance of chromosomes (SMC) family proteins condensin and cohesin Both complexes contain two long coiled-coil subunits joined at a hinge region and an ATPase head region, when bound to ATP, forming a structure with a ring topology. The physiological context of Xenopus egg extracts will be useful in determining how the cell cycle controls loop extrusion by condensin and cohesin complexes. LSS replicates Xenopus sperm chromatin [2], but this depends on nuclei formation, complicating real-time single-molecule imaging of replicating DNAs. To visualise individual DNA molecules after completion of replication in egg extracts, DNA combing and electron microscopy have been used [18]. Once licensed in HSS, diverse DNA substrates undergo a single complete round of DNA replication in NPE as origin licensing and firing are temporally separated
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