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

2019 ◽  
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.

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

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.

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

eLife ◽  
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.

scholarly journals Facilitated Dissociation of Transcription Factors from Single DNA Binding Sites

2017 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
Binding Kinetics ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Standard Picture ◽  
Dynamics Simulations

ABSTRACTThe binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits TF off-rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key E. coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∽1 × 104 M-1s-1, establishing that FD of Fis occurs at the single-binding-site level, and we find that the off-rate saturates at large Fis concentrations in solution. While spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that facilitated dissociation depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF whose structure differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.SIGNIFICANCE STATEMENTTranscription factors (TFs) control biological processes by binding and unbinding to DNA. Therefore it is crucial to understand the mechanisms that affect TF binding kinetics. Recent studies challenge the standard picture of TF binding kinetics by demonstrating cases of proteins in solution accelerating TF dissociation rates through a facilitated dissociation (FD) process. Our study shows that FD can occur at the level of single binding sites, without the action of large protein clusters or long DNA segments. Our results quantitatively support a model of FD in which competitor proteins invade partially dissociated states of DNA-bound TFs. FD is expected to be a general mechanism for modulating gene expression by altering the occupancy of TFs on the genome.Author ContributionsRamsey I. Kamardesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperEdward J. Banigandesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperAykut Erbasdesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperRebecca D. Giuntolidesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperMonica Olvera de la Cruzdesigned research, performed research, wrote the paperReid C. Johnsondesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paperJohn F. Markodesigned research, performed research, contributed new reagents/analytic tools, analyzed data, wrote the paper

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

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.

scholarly journals A Peek Inside the Machines of Bacterial Nucleotide Excision Repair

2021 ◽  
Vol 22 (2) ◽  
pp. 952
Author(s):  
Thanyalak Kraithong ◽  
Silas Hartley ◽  
David Jeruzalmi ◽  
Danaya Pakotiprapha
Keyword(s):  
Nucleotide Excision Repair ◽  
Single Molecule ◽  
Genetic Information ◽  
Excision Repair ◽  
Double Stranded Dna ◽  
Nucleotide Excision ◽  
Single Molecule Studies ◽  
Exogenous Agents ◽  
Global Genome Repair ◽  
Shed Light

Double stranded DNA (dsDNA), the repository of genetic information in bacteria, archaea and eukaryotes, exhibits a surprising instability in the intracellular environment; this fragility is exacerbated by exogenous agents, such as ultraviolet radiation. To protect themselves against the severe consequences of DNA damage, cells have evolved at least six distinct DNA repair pathways. Here, we review recent key findings of studies aimed at understanding one of these pathways: bacterial nucleotide excision repair (NER). This pathway operates in two modes: a global genome repair (GGR) pathway and a pathway that closely interfaces with transcription by RNA polymerase called transcription-coupled repair (TCR). Below, we discuss the architecture of key proteins in bacterial NER and recent biochemical, structural and single-molecule studies that shed light on the lesion recognition steps of both the GGR and the TCR sub-pathways. Although a great deal has been learned about both of these sub-pathways, several important questions, including damage discrimination, roles of ATP and the orchestration of protein binding and conformation switching, remain to be addressed.

scholarly journals Facilitated dissociation of transcription factors from single DNA binding sites

2017 ◽  
Vol 114 (16) ◽  
pp. E3251-E3257 ◽  
Author(s):  
Ramsey I. Kamar ◽  
Edward J. Banigan ◽  
Aykut Erbas ◽  
Rebecca D. Giuntoli ◽  
Monica Olvera de la Cruz ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Single Molecule ◽  
Binding Sites ◽  
General Mechanism ◽  
Dissociation Kinetics ◽  
Dna Binding Sites ◽  
Bimolecular Interactions ◽  
Dynamics Simulations ◽  
Kinetics Of

The binding of transcription factors (TFs) to DNA controls most aspects of cellular function, making the understanding of their binding kinetics imperative. The standard description of bimolecular interactions posits that TF off rates are independent of TF concentration in solution. However, recent observations have revealed that proteins in solution can accelerate the dissociation of DNA-bound proteins. To study the molecular basis of facilitated dissociation (FD), we have used single-molecule imaging to measure dissociation kinetics of Fis, a key Escherichia coli TF and major bacterial nucleoid protein, from single dsDNA binding sites. We observe a strong FD effect characterized by an exchange rate ∼1×104 M−1s−1, establishing that FD of Fis occurs at the single-binding site level, and we find that the off rate saturates at large Fis concentrations in solution. Although spontaneous (i.e., competitor-free) dissociation shows a strong salt dependence, we find that FD depends only weakly on salt. These results are quantitatively explained by a model in which partially dissociated bound proteins are susceptible to invasion by competitor proteins in solution. We also report FD of NHP6A, a yeast TF with structure that differs significantly from Fis. We further perform molecular dynamics simulations, which indicate that FD can occur for molecules that interact far more weakly than those that we have studied. Taken together, our results indicate that FD is a general mechanism assisting in the local removal of TFs from their binding sites and does not necessarily require cooperativity, clustering, or binding site overlap.

scholarly journals Characterization of the first tetrameric transcription factor of the GntR superfamily with allosteric regulation from the bacterial pathogen Agrobacterium fabrum

2020 ◽  
Author(s):  
Armelle Vigouroux ◽  
Thibault Meyer ◽  
Anaïs Naretto ◽  
Pierre Legrand ◽  
Magali Aumont-Nicaise ◽  
...  
Keyword(s):  
Binding Sites ◽  
Bacterial Pathogen ◽  
Degradation Pathway ◽  
Activity Measurement ◽  
Allosteric Mechanism ◽  
Trophic Resources ◽  
Dna Binding Sites ◽  
Species Specific ◽  
Shed Light

Abstract A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

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