Polymer-monovalent salt-induced DNA compaction studied via single-molecule microfluidic trapping

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.

Download Full-text

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

eLife ◽

10.7554/elife.64563 ◽

2021 ◽

Vol 10 ◽

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.

Download Full-text

Bridging the spatiotemporal scales of macromolecular transport in crowded biomimetic systems

10.1101/431528 ◽

2018 ◽

Cited By ~ 2

Author(s):

Kathryn Regan ◽

Devynn Wulstein ◽

Hannah Rasmussen ◽

Ryan McGorty ◽

Rae M. Robertson-Anderson

Keyword(s):

Single Molecule ◽

Biological Molecules ◽

Biological Macromolecules ◽

Dna Dynamics ◽

Dna Transport ◽

Macromolecular Transport ◽

Dna Compaction ◽

Biomimetic Systems ◽

Conformational Properties ◽

Spatiotemporal Scales

AbstractCrowding plays a key role in the transport and conformations of biological macromolecules. Gene therapy, viral infection and transfection require DNA to traverse the crowded cytoplasm, including a heterogeneous cytoskeleton of filamentous proteins. Given the complexity of cellular crowding, the dynamics of biological molecules can be highly dependent on the spatiotemporal scale probed. We present a powerful platform that spans molecular and cellular scales by coupling single-molecule conformational tracking (SMCT) and selective-plane illumination differential dynamic microscopy (SPIDDM). We elucidate the transport and conformational properties of large DNA, crowded by custom-designed networks of actin and microtubules, to link single-molecule conformations with ensemble DNA transport and cytoskeleton structure. We show that actin crowding leads to DNA compaction and suppression of fluctuations, combined with anomalous subdiffusion and heterogeneous transport, whereas microtubules have much more subdued impact across all scales. Interestingly, in composite networks of both filaments, microtubules primarily govern single-molecule DNA dynamics whereas actin governs ensemble transport.

Download Full-text

Author response: Single-molecule observation of DNA compaction by meiotic protein SYCP3

10.7554/elife.22582.013 ◽

2017 ◽

Author(s):

Johanna L Syrjänen ◽

Iddo Heller ◽

Andrea Candelli ◽

Owen R Davies ◽

Erwin J G Peterman ◽

...

Keyword(s):

Single Molecule ◽

Author Response ◽

Dna Compaction

Download Full-text

Kinetic analysis of DNA compaction by mycobacterial integration host factor at the single-molecule level

Tuberculosis ◽

10.1016/j.tube.2019.101862 ◽

2019 ◽

Vol 119 ◽

pp. 101862 ◽

Cited By ~ 1

Author(s):

Yuanyuan Chen ◽

Zhengyan Zhan ◽

Hongtai Zhang ◽

Lijun Bi ◽

Xian-En Zhang ◽

...

Keyword(s):

Kinetic Analysis ◽

Single Molecule ◽

Integration Host Factor ◽

Host Factor ◽

Single Molecule Level ◽

Dna Compaction

Download Full-text

Human cohesin compacts DNA by loop extrusion

Science ◽

10.1126/science.aaz4475 ◽

2019 ◽

Vol 366 (6471) ◽

pp. 1345-1349 ◽

Cited By ~ 128

Author(s):

Yoori Kim ◽

Zhubing Shi ◽

Hongshan Zhang ◽

Ilya J. Finkelstein ◽

Hongtao Yu

Keyword(s):

Adenosine Triphosphate ◽

Single Molecule ◽

Adenosine Triphosphatase ◽

Direct Evidence ◽

Atp Hydrolysis ◽

Average Rate ◽

Molecular Machine ◽

Single Molecule Imaging ◽

Dna Loops ◽

Dna Compaction

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

Download Full-text

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.

Download Full-text

The Major Chromosome Condensation Factors Smc, HBsu, and Gyrase in Bacillus subtilis Operate via Strikingly Different Patterns of Motion

mSphere ◽

10.1128/msphere.00817-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Sonja Schibany ◽

Rebecca Hinrichs ◽

Rogelio Hernández-Tamayo ◽

Peter L. Graumann

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Single Molecule ◽

High Mobility ◽

Single Molecule Tracking ◽

Content Type ◽

Dna Compaction ◽

Transient Interactions

ABSTRACT Although DNA-compacting proteins have been extensively characterized in vitro, knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis, Smc (structural maintenance of chromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo, avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo, displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo.

Download Full-text

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.

Download Full-text

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.

Download Full-text