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

Chen, Yuanyuan; Zhan, Zhengyan; Zhang, Hongtai; Bi, Lijun; Zhang, Xian-En; Fu, Yu

doi:10.1016/j.tube.2019.101862

Cited by 4 publications

(3 citation statements)

References 16 publications

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“…All proteins were expressed in E. coli BL21 (Biomed Inc.) with minor modifications ( 89 ). For example, Hmo1 was purified by a His-Trap HP column (binding buffer: 20 mM Tris-HCI pH 7.5, 500 mM NaCl; elution buffer: 20 mM Tris-HCI pH 7.5, 500 mM NaCl, 500 mM imidazole) and a Hi-Trap SP HP column (binding buffer: 20 mM Tris-HCI pH 6.5, 200 mM NaCl; elution buffer: 20 mM Tris-HCI pH 6.5, 1 M NaCl) using the AKTA purifier.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

Wang

et al. 2023

mBio

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Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C -terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C -terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae , even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C -terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

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Section: Methodsmentioning

confidence: 99%

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

Wang

et al. 2023

mBio

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“…Attachment sites are recognized by Int—an integral part of the attP – Int cassette required for integrase-mediated site-specific recombination ( Singh et al 2013 ). Thereby, Int is either a tyrosine recombinase (which requires additional host cofactors such as the one present in Mycobacterium smegmatis ; Pedulla et al 1996 ; Peña et al 1999 ; Lewis and Hatfull 2003 ; Chen et al 2019 ) or a serine recombinase (which functions without any cofactors but recognizes shorter attP sequences than the tyrosine recombinase; Groth and Calos 2004 ).…”

Section: Introductionmentioning

confidence: 99%

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages

Howell

Versoza

Cerna

et al. 2022

G3 Genes|Genomes|Genetics

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Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages—and thus one of the selective pressures acting on complex microbial systems in nature—remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from six sub-clusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of sub-cluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of sub-clusters P2 to P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles—a temperate characteristic that impedes their usage in anti-bacterial applications.

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“…These natural condensation processes have attracted significant interest not only in polymer physics but also in pharmaceutical applications in gene delivery systems. Various artificial condensing agents have been explored, such as basic proteins, , alcohols, , multivalent cations, , neutral molecular crowding polymers, − and their combination. , The development of single-molecule techniques including optical tweezers, atomic force microscopy (AFM), , and microfluidic devices ,− has enabled the investigation of the semiflexible properties of DNA molecules under external forces (optical radiation, mechanical, magnetic, or hydrodynamic) through the condensation and decondensation processes. Although the ethanol precipitation technique based on the above condensation and decondensation phenomenon is widely used in DNA purification, and the statistical structures have been well-studied, the kinetics of the DNA conformational transition induced by ethanol at the single-molecule level has not been thoroughly understood.…”

mentioning

confidence: 99%

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System

et al. 2020

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Conformational transitions from secondary (e.g., B-to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several singlemolecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil−globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

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

Cited by 4 publications

References 16 publications

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages

Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System

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