Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes

The data presented in this article are related to the research article entitled "Quantitative determination of linking number differences between circular polynucleosomes and histone H1-bound circular polynucleosomes" Zhang et al. (in press) [1]. DNA linking number differences between histone H1-free and histone H1-bound circular polynucleosomes at various spermidine concentrations was quantitatively determined by chloroquine-based gel electrophoretic analysis in this work, which provides information on the topological effects of histone H1 and spermidine on the linker DNA between nucleosomes.


Data format
Raw, analyzed Experimental factors The histones, enzymes and plasmid DNA were purchased from New England Biolabs Inc. Polynucleosomes were prepared following the Dilution Assembly Protocol (E5350) provided by New England Biolabs Inc. Experimental features DNA linking number distribution was determined by agarose electrophoresis at 1.5 V/cm in 1× TAE buffer for 12 h in the presence of 1.2 μg/ml of chloroquine.

Data source location
Nanyang Technological University, Singapore Data accessibility Data within this and the main article.

Value of the data
Contribution of histone H1 to DNA linking number is quantitatively measured, which could be valuable for elucidation of the roles of linker histones in spatial arrangement of chromatin.
Concentration-dependent effects of spermidine on DNA linking number of polynucleosomes is of value for the experimental design in the future biochemical and biomedical research.
The chloroquine-based electrophoresis method could be useful for determination of DNA linking number differences and analysis of DNA topoisomer distributions.

Data
It has been reported in Ref. [1] that binding of histone H1 molecules produces linking number changes in circular polynucleosomes in the presence of 1.5 mM spermidine [1]. With the aim of knowing the effects of spermidine concentration on the linking number changes caused by histone H1, various amounts of spermidine (0 mM, 0.75 mM, 2 mM, 3 mM, 5 mM) were incubated with the open-circular polynucleosomes respectively before ligation reactions. The distribution patterns of these obtained DNA topoisomers were subsequently resolved by chloroquine-based gel electrophoresis (data presented in Figs. [1][2][3][4][5]. In addition, effects of spermidine alone (without involvement of histone H1) on the linking number of circular polynucleosomes were determined and presented in Fig. 6 in this article as well.

Construction of open-circular polynucleosomes
Preparation of open-circular histone H1-free polynucleosome (Structure 3) and open-circular histone H1-bound polynucleosome (Structure 4) was carried out though the following procedures which has been described in Ref. [1] as well: Step 1: One of the DNA strands was cleaved at a specific recognition sequence by the action of DNA nicking endonuclease. 5 μg pBR322 (Structure 1) was cleaved by 5 U of Nt.BspQI nicking endonuclease at 50°C over 2 hours in a 50 μl buffer solution (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl 2 , 1 mM DTT). The obtained reaction products (Structure 2) were subsequently purified by QIAquick PCR Purification Kit.  Fig. 1e, which gave rise to − 6.07 as its mean value of ΔLk.  Fig. 2e, which gave rise to − 5.98 as its mean value of ΔLk.
Step 2: Open-circular polynucleosomes (Structure 3) were constructed following the manufacturer's protocol and our previous experimental procedures [2,3]. The open-circular plasmid DNA was incubated with histone H2A/H2B dimer and histone H3.1/H4 tetramer in the presence of 2 M NaCl. This 10 μl mixture was then stepwisely diluted with 10 mM Tris-HCl buffer (pH 8.0) to final volume of 400 μl.

Construction of close circular polynucleosomes
Open-circular polynucleosomes (Structure 3 or Structure 4) were incubated with certain amount of spermidine at room temperature for 30 min. 100 units of T4 DNA ligase were then incubated with these open-circular polynucleosomes at room temperature for 2 hours in the presence of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM ATP and 10 mM DTT. The reaction products were treated with 10 units of Proteinase K for 30 min. After removal of proteins by QIAquick PCR Purification Kit, the circular DNA were then incubated with 30 units of Escherichia coli Exonuclease III in the presence of 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 and 1 mM DTT at 37°C for 30 min before gel electrophoretic analysis.

Determination of DNA linking number changes by gel electrophoresis
Electrophoretic examinations were carried out on 1.4% agarose gel (with 1.2 μg/ml chloroquine inside the gel) at 1.5 V/cm. Gel was stained with ethidium bromide and subsequently visualized using    Fig. 5e, which gave rise to − 5.32 as its mean value of ΔLk.