Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts

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.

Tools for Decoding Ubiquitin Signaling in DNA Repair

“…The control and reproducibility of this system allows experimental design with high spatial and temporal resolution, with typical assay outputs varying from immunoblot analysis of chromatin extracts over a particular time course, targeted enrichment of a particular protein of interest, or mass spectrometry for protein identification and/or analysis of PTMs in response to a particular DNA lesion ( Gallina et al, 2021 ). Moreover, in recent years, this cell-free extract system has also been coupled with single-molecule techniques ( Gruszka et al, 2020 ; Cameron and Yardimci, 2021 ). Lastly, to more accurately define the DDR and ubiquitin signaling in response to particular DNA lesions, specifically designed DNA plasmid templates can be used with the Xenopus egg extracts ( Hoogenboom et al, 2017 ).…”

Biochemistry: one molecule at a time

“…single-ion channel recordings using patch-clamp [1], the single-molecule toolkit has expanded considerably and continues to evolve. Single-molecule detection and manipulation techniques can broadly be divided into the following categories: (i) light microscopy approaches, including fluorescence imaging and spectroscopy [2][3][4][5][6][7], and interferometric scattering [8], (ii) electrical conductance measurements, including patch-clamp and nanopore-based detection [9,10], and (iii) force-based approaches, including optical [11] and magnetic tweezers [12], and atomic force microscopy [13]. Different single-molecule methods have different capabilities, applications and throughput, and offer different time and spatial resolutions.…”

“…As the temporal and spatial resolutions of single-molecule methods have been improving over the years, the complexity of analysed samples has also increased, opening up new avenues for scientific discovery. Single-molecule studies within cells [7] or cell extracts [3] are now routinely conducted, in addition to in vitro assays using purified components. The in vivo single molecule approaches mostly involve fluorescence detection [19][20][21][22][23] however force-based technologies are also being developed [24,25].…”