Cameron G and Yardimci H (2021), Studying chromosome biology with single-molecul...

XB-ART-57721

Essays Biochem 2021 Apr 16;651:17-26. doi: 10.1042/EBC20200026.

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

Cameron G , Yardimci H .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cdc6 cdt1 fen1 mcm2 tdp1

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	Figure 1. Single-molecule techniques to investigate chromosome organisation(A) An optical trap measuring forces involved in chromatinisation of λ DNA in Xenopus egg extracts [6]. λ DNA attached to beads at both ends is bound at one end to a micropipette, then incubated in extracts to form chromatin. After chromatin assembly, extract is replaced with buffer and the second bead optically trapped. The micropipette is moved away from the optical trap, and force in DNA fibre is continuously measured, shown here in the force extension curve. (B) Adding nucleosome-depleted egg extracts to λ DNA double-tethered to the surface of a coverslip allows loop extrusion to occur. Loops appear as regions of high SYTOX staining on DNA, visualised by TIRF microscopy, unless applying perpendicular flow [15]. Abbreviation: TIRF, total internal reflection fluorescence.
	Figure 2. DNA replication in X. laevis extracts(A) During G1-phase, DNA is licensed at origins of replication. The ORC, Cdc6 and Cdt1 load Mcm2-7 helicases as inactive DH complexes around dsDNA. HSS extracts license DNAs with DHs, with ORC in HSS binding DNA in a sequence-independent manner, so specific origins are not required and different DNAs can be licensed in HSS [51]. (B) In S-phase, a number of essential firing factors initiate DNA replication. Mcm2-7 from DHs is remodelled into the CMG helicase which binds ssDNA and unwinds DNA in the 3′-to-5′ direction. Other replisome components assemble around CMG [52]. The events of origin firing are recapitulated by adding NPE to DNAs licensed in HSS.
	Figure 3. Single-molecule techniques to study DNA replication in extracts(A) λ DNA can be replicated in a microfluidic flow cell using a licensing mix, containing HSS, followed by a replication mix, containing HSS and NPE. Adding a second replication mix containing the CDK inhibitor p27kip prevents excessive origin firing. Digoxigenin-dUTP labels nascent DNA, which can be immunostained [22]. (B) Real-time imaging of replication forks in extracts with Fen1-mKikGR, which binds nascent DNA [25], and adaptation of this system to visualise the fate of labelled nucleosomes reconstituted on λ DNA [26]. The right hand panel shows a kymogram, a stack of images from a single position of interest, with a growing replication bubble marked by Fen1-mKikGR. After the right hand replication fork reaches the labelled nucleosome four different histone fates are observed. (C) Summary of the KERHMIT assay developed in [33]. The bottom panel shows an example kymogram, where the right hand replication fork collides with a stable leading strand DPC. After initial pausing at the DPC, CMG is shown to bypass the DPC, with the fork rate slowing down until possible recoupling to polymerase. Abbreviation: DPC, DNA–protein cross-link.
	Figure 4. Studying the mechanism of NHEJ by smFRET(A) Intermolecular end joining assay for monitoring NHEJ in HSS. The Cy3 donor fluorophore is attached to a 100-bp DNA on the surface of a coverslip inside a microfluidic flow cell. Cy5 association with Cy3 with no FRET shows long range complex formation, followed by conversion into short range complexes, where FRET between the fluorophore pair occurs [40]. (B) Intramolecular end joining assay, where Cy3 and Cy5 fluorophores are at the ends of a DNA fragment bound to the surface with an internal biotin [40]. Cy3 and Cy5 continuously are present in a diffraction-limited spot but only show FRET when a short-range complex forms. (C) Modifications to intramolecular end joining assays for monitoring enzymatic processing of non-compatible DNA ends [42]. (i) pol λ fills gaps in substrates with resected 3′ DNA ends, detected here by incorporation of a nucleotide modified with BHQ, which quenches Cy3 fluorescence. (ii) Tdp1 removes adducts on 3′ DNA ends, in this example removing Cy3 modification at one 3′ DNA end.

References [+] :

Barrows, Cell-free transcription in Xenopus egg extract. 2019, Pubmed, Xenbase