Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Summary

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Abstract

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

Introduction

Eukaryotic DNA is packaged into a higher-order structure known as chromatin^1,2. Nucleosome is the fundamental unit of chromatin, which consists of approximately 147 bp DNA wrapped around the octameric core histones^3,4. Chromatin plays a critical role in eukaryotic cells; for example, the compact structure protects DNA from endogenous factors and exogenous threats⁵. Chromatin structure changes dynamically according to the cell cycle phase and environmental stimuli, and these changes control protein access during DNA transactions ....

Protocol

1. Preparation of the flow cell

1. Prepare a cleaned fused silica slide containing nano-trench patterns following previously published report²⁵.
   1. Drill two holes with 1 mm diameter in a cleaned fused silica slide (Figure 1A) using a diamond-coated drill bit (see Table of Materials).
   2. Deposit 250 nm of thick aluminum (Al) on the slide using DC sputter³² (see Table of Materia.......

Discussion

As a single-molecule imaging technique, DNA curtain has been used extensively to probe DNA metabolic reactions⁴³. DNA curtain is a hybrid system that concatenates lipid fluidity, microfluidics, and TIRFM. Unlike other single-molecule techniques, DNA curtain enables high-throughput real-time visualization of protein-DNA interactions. Therefore, the DNA curtain technique is suitable for probing the mechanism behind molecular interactions, including sequence-specific association, protein movement alo.......

Materials

Name	Company	Catalog Number	Comments
1 mL luer-lock syringe	BecktonDickinson	301321
1' x 3' fused-silca slide glass	G. Finkenbeiner		1 inch x 3 inch rectangular and 1 mm thickness
10 mL luer-lock syringe	BecktonDickinson	302149
18:1 (Δ9-Cis) PC (DOPC)	Avanti	850375	This is a component of biotinylated lipid stock
18:1 Biotinyl cap PE	Avanti	870273	This is a component of biotinylated lipid stock
18:1 PEG2000 PE	Avanti	880130	This is a component of biotinylated lipid stock
3 mL luer-lock syringe	BecktonDickinson	302832
6-way sample injection valve	IDEX	MX series II
950K PMMA	All-resist	671.04
Acetone	SAMCHUN	A1759
Adenosine 5'-triphosphate disodium salt hydrate (ATP)	Sigma	A2383
Aluminum (Al)	TASCO, South Korea	LT50AI414	Diameter 4 inch, thickness 1/4 inch
Amicon Ultra centrifugal filter, MWCO 10 kDa	Millipore	Z648027
Ampicillin	Mbcell	MB-A4128	Antibiotics
AZ 300 MIF developer	Merck	10454110521	Used for removing aluminum
Blade	DORCO	DN52	12 mm x 6 m
Boron trichloride (BCl3)	UNIONGAS		Purity: >99.99%
Bovine serum albumin (BSA)	Sigma	A7030
Catalase	Sigma	C40-1g	This is a component of 100x gloxy stock
Chlorine (Cl2)	UNIONGAS		Purity: >99.99%
Clear double-sided tape	3M	313770
D-(+)-glucose	Sigma	G7528
DC sputter	Sorona	SRM-120	Used for deposition aluminum on a slide
Diamond-coated drill bit	Eurotool	DIB-211.00	Used for making holes in a fusced silica slide
DL-Dithiothreitol (DTT)	Sigma	D0632
Dove-prism	Korea Electro-Optics Co. Ltd.	1906-106	Custom-made fused-silica dove prism with anti-reflection coating
Drill	Dremel	Dremel 3000	Used for making holes in a fusced silica slide
Electron Bean Lithography	Nanobeam Ltd.	NB3
Ethylene-diamine-tetraacetic acid (EDTA)	Sigma	EDS-1KG
Fingertight fittings	IDEX	F-300	It is connected with "PFA Tubing Natural" to form luer-lock tubing
Flangeless male nut	IDEX	P-235	It is connected with "PFA Tubing Natural" to form luer-lock tubing
Freeze Dryer, HyperCOOL	Labogene	HC3110	Used for lyophilizing liquid proteins
Glucose oxidase	Sigma	G2133-50KU	This is a component of 100x gloxy stock
Guanidinium hydrochloride	Acros Organics	364790025
Hamilton syringe	Hamilton Company	80065	This syringe is used for sample injection
Hellmanex III	Sigma	Z805939
HiLoad 26/600 SuperdexTM 200 pg	Cytiva	28-9893-36	Used for FPLC (size exclusion)
Hot plate stirrer	Corning	PC-420D
Hydrochloric acid	Sigma	H1759	Used for Tris-HCl
Index matching oil	ZEISS	444970-9000-000
Inductively coupled plasma-reactive ion etching	Top Technology Ltd.	FabStar
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Glentham Life Sciences	GC6586-100g	Used for induction of β-galactosidase activity
Lambda phage DNA	NEB	N0311
LB broth	BD difco	244610	Media for E.coli cell growth
Luer adapter 10-32	IDEX	P-659	This connects luer-lock syringe and tubing
Magnesium chloride hexahydrate	fisher bioreagents	BP214
Methyl isobutyl ketone (MIBK)	KAYAKU ADVANCED MATERIALS		Used for developing solution
Microscope (Eclipse Ti2)	Nikon	Eclipse Ti2	Inverted fluorescence microscope
Microscope glass coverslip	MARIENFELD	101142	22 x 50 mm (No. 1)
Microscope slide	DURAN GROUP	DU.2355013	Slide glass ground edge 45°, plain 26 x 76 mm
Nanoport	IDEX	N-333-01
Objective lens	Nikon	CFI Plan Apochromat VC 60XC WI	Immersion type: water, magnification: 60x, correction: 18, working distance: 0.29 (0.31-0.28)
One Shot BL21 (DE3)pLysS Chemically Competent E. coli	Thermo Fisher Scientific	C6060-03	Competent cell for overexpressing proteins
Oxygen (O2)	NOBLEGAS, South Korea		Purity: >99.99%
PFA tubing natural	IDEX	1512L	It is connected with "Fingertight Fittings" to form luer-lock tubing
Phenylmethylsulfonyl fluoride (PMSF)	Roche	11359061001	Protease inhibitor
Sephacryl S-200 High Resolution	Cytiva	17-0584-01	Used for FPLC (size exclusion)
Shut-off valve	IDEX	P-732
Sodium acetate	Sigma	791741
Sodium chloride (NaCl)	Sigma	S3014
Sodium hydroxide (NaOH)	Sigma	s5881
Spectra/Por molecularporous membrane tubing, MWCO 6-8 kDa	Spectrum laboratories	132660
Streptavidin	Thermo Fisher Scientific	S888
Sulfur tetralfluoride (SF4)	NOBLEGAS, South Korea		Purity: >99.99%
Syringe pump	KD Scientific	78-8210
Tetrafluoromethane (CF4)	NOBLEGAS, South Korea		Purity: >99.99%
TritonX-100	Sigma	T9284
Trizma base	Sigma	T1503	Used for Tris-HCl
TSKgel SP-5PW	TOSOH	14715	Used for FPLC (ion exchange)
Union assembly	IDEX	P-760	This connects tubings
Urea	Sigma	U5378
Vacuum oven	Jeio Tech	OV-11
YOYO-1	Thermo Fisher Scientific	Y3601	This intercalation dye is diluted in DMSO
β-mercaptoethanol (BME)	Sigma	M6250