scholarly journals Archaeal Chromatin Proteins Cren7 and Sul7d Compact DNA by Bending and Bridging

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Zhenfeng Zhang ◽  
Zhengyan Zhan ◽  
Bing Wang ◽  
Yuanyuan Chen ◽  
Xiuqiang Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Bending ◽  
Chromosomal Dna ◽  
Content Type ◽  
Single Molecule Level ◽  
Dna Compaction ◽  
Dna Organization ◽  
Chromatin Proteins ◽  
Mechanism And Kinetics ◽  
Kinetics Of

ABSTRACT Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered λ DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea. IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

Revealing Kinetics of Two-Electron Oxygen Reduction Reaction at Single-Molecule Level

2020 ◽  
Vol 142 (30) ◽  
pp. 13201-13209
Author(s):  
Yi Xiao ◽  
Jaeyoung Hong ◽  
Xiao Wang ◽  
Tao Chen ◽  
Taeghwan Hyeon ◽  
...  
Keyword(s):  
Oxygen Reduction Reaction ◽  
Oxygen Reduction ◽  
Single Molecule ◽  
Reduction Reaction ◽  
Single Molecule Level ◽  
Kinetics Of

scholarly journals Nuclease dead Cas9 is a programmable roadblock for DNA replication

2018 ◽  
Author(s):  
Kelsey Whinn ◽  
Gurleen Kaur ◽  
Jacob S. Lewis ◽  
Grant Schauer ◽  
Stefan Müller ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Molecular Machines ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Single Molecule Level ◽  
Replication Fork Arrest

DNA replication occurs on chromosomal DNA while processes such as DNA repair, recombination and transcription continue. However, we have limited experimental tools to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct fused to the photo-stable dL5 protein fluoromodule as a novel, targetable protein-DNA roadblock for studying replication fork arrest at the single-molecule level in vitro as well as in vivo. We find that the specifically bound dCas9–guideRNA complex arrests viral, bacterial and eukaryotic replication forks in vitro.

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

Tuberculosis ◽  
2019 ◽  
Vol 119 ◽  
pp. 101862 ◽  
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

scholarly journals Controlling load-dependent kinetics of β-cardiac myosin at the single-molecule level

2018 ◽  
Vol 25 (6) ◽  
pp. 505-514 ◽  
Author(s):  
Chao Liu ◽  
Masataka Kawana ◽  
Dan Song ◽  
Kathleen M. Ruppel ◽  
James A. Spudich
Keyword(s):  
Single Molecule ◽  
Cardiac Myosin ◽  
Single Molecule Level ◽  
Kinetics Of

scholarly journals Dissecting the self-assembly kinetics of multimeric pore-forming toxins

2016 ◽  
Vol 13 (114) ◽  
pp. 20150762 ◽  
Author(s):  
A. A. Lee ◽  
M. J. Senior ◽  
M. I. Wallace ◽  
T. E. Woolley ◽  
I. M. Griffiths
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Quantitative Agreement ◽  
Time Resolved ◽  
Biologically Relevant ◽  
Low Concentrations ◽  
Assembly Kinetics ◽  
Quantitative Understanding ◽  
Mechanism And Kinetics ◽  
Kinetics Of

Pore-forming toxins are ubiquitous cytotoxins that are exploited by both bacteria and the immune response of eukaryotes. These toxins kill cells by assembling large multimeric pores on the cell membrane. However, a quantitative understanding of the mechanism and kinetics of this self-assembly process is lacking. We propose an analytically solvable kinetic model for stepwise, reversible oligomerization. In biologically relevant limits, we obtain simple algebraic expressions for the rate of pore formation, as well as for the concentration of pores as a function of time. Quantitative agreement is obtained between our model and time-resolved kinetic experiments of Bacillus thuringiensis Cry1Ac (tetrameric pore), aerolysin, Staphylococcus aureus α -haemolysin (heptameric pores) and Escherichia coli cytolysin A (dodecameric pore). Furthermore, our model explains how rapid self-assembly can take place with low concentrations of oligomeric intermediates, as observed in recent single-molecule fluorescence experiments of α-haemolysin self-assembly. We propose that suppressing the concentration of oligomeric intermediates may be the key to reliable, error-free, self-assembly of pores.

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

mSphere ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document