Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts.
Cameron G
,
Yardimci H
.
???displayArticle.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.
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 righthand 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 righthand 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 righthand 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.
Barrows,
Cell-free transcription in Xenopus egg extract.
2019, Pubmed,
Xenbase
Barrows,
Cell-free transcription in Xenopus egg extract.
2019,
Pubmed
,
Xenbase
Bell,
Chromosome Duplication in Saccharomyces cerevisiae.
2016,
Pubmed
Bennink,
Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers.
2001,
Pubmed
,
Xenbase
Blow,
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
1986,
Pubmed
,
Xenbase
Davidson,
DNA loop extrusion by human cohesin.
2019,
Pubmed
Di Virgilio,
Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11.
2005,
Pubmed
,
Xenbase
Duxin,
Repair of a DNA-protein crosslink by replication-coupled proteolysis.
2014,
Pubmed
,
Xenbase
Escobar,
Active and Repressed Chromatin Domains Exhibit Distinct Nucleosome Segregation during DNA Replication.
2019,
Pubmed
Fu,
Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.
2011,
Pubmed
,
Xenbase
Ganji,
Real-time imaging of DNA loop extrusion by condensin.
2018,
Pubmed
Gillespie,
Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.
2012,
Pubmed
,
Xenbase
Golfier,
Cohesin and condensin extrude DNA loops in a cell cycle-dependent manner.
2020,
Pubmed
,
Xenbase
Graham,
A single XLF dimer bridges DNA ends during nonhomologous end joining.
2018,
Pubmed
,
Xenbase
Graham,
Two-Stage Synapsis of DNA Ends during Non-homologous End Joining.
2016,
Pubmed
Gruszka,
Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication.
2020,
Pubmed
,
Xenbase
Herrick,
Replication fork density increases during DNA synthesis in X. laevis egg extracts.
2000,
Pubmed
,
Xenbase
Hirano,
A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.
1994,
Pubmed
,
Xenbase
Hoogenboom,
Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.
2017,
Pubmed
,
Xenbase
Kanke,
Cohesin acetylation and Wapl-Pds5 oppositely regulate translocation of cohesin along DNA.
2016,
Pubmed
,
Xenbase
Kim,
Human cohesin compacts DNA by loop extrusion.
2019,
Pubmed
Kose,
Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model.
2019,
Pubmed
,
Xenbase
Kurat,
Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.
2017,
Pubmed
Ladoux,
Fast kinetics of chromatin assembly revealed by single-molecule videomicroscopy and scanning force microscopy.
2000,
Pubmed
,
Xenbase
Laskey,
Assembly of SV40 chromatin in a cell-free system from Xenopus eggs.
1977,
Pubmed
,
Xenbase
Lebofsky,
DNA replication in nucleus-free Xenopus egg extracts.
2009,
Pubmed
,
Xenbase
Lewis,
Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication.
2020,
Pubmed
Loveland,
A general approach to break the concentration barrier in single-molecule imaging.
2012,
Pubmed
,
Xenbase
Low,
The DNA replication fork suppresses CMG unloading from chromatin before termination.
2020,
Pubmed
,
Xenbase
Marheineke,
Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.
2004,
Pubmed
,
Xenbase
Marheineke,
Use of DNA combing to study DNA replication in Xenopus and human cell-free systems.
2009,
Pubmed
,
Xenbase
Murayama,
DNA Entry into and Exit out of the Cohesin Ring by an Interlocking Gate Mechanism.
2015,
Pubmed
Murayama,
Establishment of DNA-DNA Interactions by the Cohesin Ring.
2018,
Pubmed
Murray,
Cell cycle extracts.
1991,
Pubmed
Newport,
A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage.
1982,
Pubmed
,
Xenbase
Scully,
DNA double-strand break repair-pathway choice in somatic mammalian cells.
2019,
Pubmed
Shintomi,
Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts.
2017,
Pubmed
,
Xenbase
Sparks,
The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair.
2019,
Pubmed
,
Xenbase
Stingele,
Mechanisms of DNA-protein crosslink repair.
2017,
Pubmed
Stinson,
A Mechanism to Minimize Errors during Non-homologous End Joining.
2020,
Pubmed
,
Xenbase
Takahashi,
Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex.
2004,
Pubmed
,
Xenbase
Taylor,
Dynamics of Replication Fork Progression Following Helicase-Polymerase Uncoupling in Eukaryotes.
2019,
Pubmed
Uhlmann,
SMC complexes: from DNA to chromosomes.
2016,
Pubmed
Vrtis,
Single-strand DNA breaks cause replisome disassembly.
2021,
Pubmed
,
Xenbase
Walter,
Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
2000,
Pubmed
,
Xenbase
Walter,
Regulated chromosomal DNA replication in the absence of a nucleus.
1998,
Pubmed
,
Xenbase
Wang,
Dissection of DNA double-strand-break repair using novel single-molecule forceps.
2018,
Pubmed
Yardimci,
Single-molecule analysis of DNA replication in Xenopus egg extracts.
2012,
Pubmed
,
Xenbase
Yardimci,
Uncoupling of sister replisomes during eukaryotic DNA replication.
2010,
Pubmed
,
Xenbase
Yatskevich,
Organization of Chromosomal DNA by SMC Complexes.
2019,
Pubmed
Yeeles,
Regulated eukaryotic DNA replication origin firing with purified proteins.
2015,
Pubmed
Zhao,
The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis.
2019,
Pubmed