HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

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HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

10.1101/2020.10.30.362772 ◽

2020 ◽

Author(s):

Madeline M. Keenen ◽

David Brown ◽

Lucy D. Brennan ◽

Roman Renger ◽

Harrison Khoo ◽

...

Keyword(s):

Phase Separation ◽

Single Molecule ◽

Genome Organization ◽

Stable Phase ◽

Dna Molecules ◽

Weakly Bound ◽

Dna Compaction ◽

Polymer Properties ◽

Molecular Scale ◽

Hp1 Proteins

In mammals HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

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Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

10.1101/149138 ◽

2017 ◽

Cited By ~ 3

Author(s):

Jorine M. Eeftens ◽

Shveta Bisht ◽

Jacob Kerssemakers ◽

Christian H. Haering ◽

Cees Dekker

Keyword(s):

Real Time ◽

Single Molecule ◽

Electrostatic Interactions ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Magnetic Tweezers ◽

Condensed State ◽

Binding Modes ◽

Dna Molecules ◽

Dna Compaction

ABSTRACTCondensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

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Human condensin I and II drive extensive ATP–dependent compaction of nucleosome–bound DNA

10.1101/683540 ◽

2019 ◽

Cited By ~ 9

Author(s):

Muwen Kong ◽

Erin Cutts ◽

Dongqing Pan ◽

Fabienne Beuron ◽

Thangavelu Kaliyappan ◽

...

Keyword(s):

Motor Activity ◽

Single Molecule ◽

Binding Sites ◽

Genome Organization ◽

Mechanisms Of Action ◽

Dna Compaction ◽

Double Stranded Dna ◽

Dna Binding Sites ◽

Structural Maintenance Of Chromosomes ◽

Shed Light

AbstractStructural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA–binding sites. Using single–molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double–stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome fibers. These observations shed light on critical processes involved in genome organization in human cells.One Sentence SummaryATP–dependent DNA compaction by human condensin complexes.

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Shelterin components modulate nucleic acids condensation and phase separation

10.1101/2021.04.30.442189 ◽

2021 ◽

Author(s):

Andrea Soranno ◽

J. Jeremías Incicco ◽

Paolo De Bona ◽

Eric J. Tomko ◽

Eric A. Galburt ◽

...

Keyword(s):

Phase Separation ◽

Single Molecule ◽

Finite Size ◽

Protein Composition ◽

Nuclear Bodies ◽

Scaffolding Protein ◽

Single Chain ◽

Dna Compaction ◽

Pml Nuclear Bodies ◽

The Relationship

AbstractTelomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, upon changes in their protein composition, telomeres can cluster to form telomere-induced-foci (TIFs) or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

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Single bacterial resolvases first exploit, then constrain intrinsic dynamics of the Holliday junction to direct recombination

Nucleic Acids Research ◽

10.1093/nar/gkab096 ◽

2021 ◽

Author(s):

Sujay Ray ◽

Nibedita Pal ◽

Nils G Walter

Keyword(s):

Cluster Analysis ◽

Homologous Recombination ◽

Single Molecule ◽

Holliday Junction ◽

Energetic Cost ◽

Genomic Integrity ◽

Dna Molecules ◽

Single Molecule Fluorescence ◽

And Cluster Analysis ◽

Fluorescence Observation

Abstract Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by ‘snap-locking’ of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.

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A Personal Journey across Fluorescent Sensing and Logic Associated with Polymers of Various Kinds

Polymers ◽

10.3390/polym11081351 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1351 ◽

Cited By ~ 4

Author(s):

Chao-Yi Yao ◽

Seiichi Uchiyama ◽

A. Prasanna de Silva

Keyword(s):

Phase Separation ◽

Metal Ion ◽

Chemical Species ◽

Polymer Science ◽

Molecular Sensing ◽

Fluorescent Sensing ◽

Molecular Logic ◽

Fluorescent Molecular Sensing ◽

Molecular Scale ◽

Identification Tags

Our experiences concerning fluorescent molecular sensing and logic devices and their intersections with polymer science are the foci of this brief review. Proton-, metal ion- and polarity-responsive cases of these devices are placed in polymeric micro- or nano-environments, some of which involve phase separation. This leads to mapping of chemical species on the nanoscale. These devices also take advantage of thermal properties of some polymers in water in order to reincarnate themselves as thermometers. When the phase separation leads to particles, the latter can be labelled with identification tags based on molecular logic. Such particles also give rise to reusable sensors, although molecular-scale resolution is sacrificed in the process. Polymeric nano-environments also help to organize rather complex molecular logic systems from their simple components. Overall, our little experiences suggest that researchers in sensing and logic would benefit if they assimilate polymer concepts.

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Molecular Transport Junctions: An Introduction

MRS Bulletin ◽

10.1557/mrs2004.119 ◽

2004 ◽

Vol 29 (6) ◽

pp. 376-384 ◽

Cited By ~ 29

Author(s):

Cherie R. Kagan ◽

Mark A. Ratner

Keyword(s):

Thin Film ◽

Charge Transport ◽

Single Molecule ◽

Molecular Transport ◽

Molecular Junction ◽

Theoretical Understanding ◽

Future Directions ◽

Molecular Scale ◽

Molecular Charge ◽

Made In

AbstractThis issue of MRS Bulletin on molecular transport junctions highlights the current experimental and theoretical understanding of molecular charge transport and its extension to the rapidly growing areas of molecular and carbon nanotube electronics. This introduction will outline the progress that has been made in understanding the mechanisms of molecular junction transport and the challenges and future directions in exploring charge transport on the molecular scale. In spite of the substantial challenges, molecular charge transport is of great interest for its intrinsic importance to potential single-molecule electronic, thin-film electronic, and optoelectronic applications.

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DNA sliding and loop formation by E. coli SMC complex: MukBEF

10.1101/2021.07.17.452765 ◽

2021 ◽

Author(s):

man zhou

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Dna Interaction ◽

Loop Formation ◽

Unknown Mechanism ◽

E Coli ◽

Dna Compaction ◽

And Function ◽

Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

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Somatic mutation landscapes at single-molecule resolution

10.21203/rs.3.pex-1298/v1 ◽

2021 ◽

Author(s):

Stefanie V. Lensing ◽

Peter Ellis ◽

Federico Abascal ◽

Iñigo Martincorena ◽

Robert J. Osborne

Keyword(s):

Single Molecule ◽

Single Cells ◽

Error Rates ◽

Dna Molecules ◽

Base Pairs ◽

Single Dna Molecules ◽

Sequencing Library ◽

Somatic Mutagenesis ◽

Qpcr Quantification ◽

Duplex Sequencing

Abstract Somatic mutations drive cancer development and may contribute to ageing and other diseases. Yet, the difficulty of detecting mutations present only in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. To overcome these limitations, we introduce nanorate sequencing (NanoSeq), a new duplex sequencing protocol with error rates <5 errors per billion base pairs in single DNA molecules from cell populations. The version of the protocol described here uses clean genome fragmentation with a restriction enzyme to prevent end-repair-associated errors and ddBTPs/dATPs during A-tailing to prevent nick extension. Both changes reduce the error rate of standard duplex sequencing protocols by preventing the fixation of DNA damage into both strands of DNA molecules during library preparation. We also use qPCR quantification of the library prior to amplification to optimise the complexity of the sequencing library given the desired sequencing coverage, maximising duplex coverage. The sample preparation protocol takes between 1 and 2 days, depending on the number of samples processed. The bioinformatic protocol is described in:https://github.com/cancerit/NanoSeqhttps://github.com/fa8sanger/NanoSeq_Paper_Code

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