Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Podsumowanie

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Streszczenie

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Wprowadzenie

In eukaryotic organisms, genome duplication is initiated from multiple sites called replication origins, where the origin recognition complex (ORC) binds and initiates the recruitment protein machinery, including the CMG helicase, to initiate DNA replication¹. The replisome machinery at each replication origin forms two replication forks that move bidirectionally^2,3. The twin replication forks replicate the two parent strands of DNA through the continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand. Given this asymmetric nature of each replication f....

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Protokół

NOTE: For buffer and reagent preparation guidelines, see buffer and reagent preparation guidelines (Supplementary File 1).

1. Histone eSPAN in budding yeast

NOTE: Most yeast strains lack a functional nucleoside transporter for thymidine uptake. To efficiently label DNA with BrdU or EdU for eSPAN, a thymidine kinase-positive (TK+) strain²¹ is required. Additionally, only mating type 'A' yeast strains can be used for cell cycle arrest using alpha factor.

1. Yeast cell synchronization and BrdU labeling (8 h)
   1. Inoculate a single col....

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Wyniki

eSPAN can be utilized to acquire strand-specific information regarding a target protein, such as histones and histone modifications. In this representative result, sample collection and eSPAN processes were conducted following the schematic outlined in Figure 2 using the budding yeast system. The sample treatment and collection follow the hydroxyurea treated early S phase condition as this protocol. Two yeast strains, WT (wild-type) and mcm2-3A mutant, were employed. The DNA helicas.......

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Dyskusje

Detection of proteins associated with leading and lagging replication strands is important for understanding the mechanisms of chromatin replication and the associated process, which are directly relevant to human cancer research and the mechanisms of drug resistance. Several methods, such as 2D gel, native Chromatin immunoprecipitation(nChIP), iPOND, and nascent chromatin capture^29,30,31, have been employed to study DNA replica.......

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Materiały

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	15575020
1.5 mL DNA low-binding tubes	Eppendorf	22431021
10x Ampligase buffer	Lucigen	A1905B
10x PBS	Corning	21-040-CV
10x TBS	BioRad	1706435
16 G x 1-1/2 in. BD PrecisionGlid Needle	BD Biosciences	305198
ACCEL-NGS 1S PLUS DNA LIBRARY KIT	Integrated DNA Technologies	10009817	ssDNA and low-input DNA library prep kit
Agilent 2100 bioanalyzer	Agilent	2100 bioanalyze
Agilent DNA 1000 kit	Agilent	5607-1504
Ampligase	Lucigen	A0110K	Handle paraformaldehyde with care, it is flammable solid hazardous.
AMPure XP	Beckman	A63881
AMPure XP SPRI Reagent	Beckman Coulter	A63882
Anti-BrdU antibody	BD Biosciences	555627
Anti-H3K4me3 antibody	abcam	ab8580
Automated cell counter , model TC10	BioRad	145-0001
Bacitracin	Sigma	11702
Bacto peptone	Thermo Fisher Scientific	211830
Benzamidine Hydrochloride	Sigma	B2417	Combustible Solids
BrdU	Sigma	B5002	Handle BrdU with care as it is a potential mutagen and teratogen.
BSA	New England Biolabs	B9000S
CaCl2	Sigma	C4901
Cell culture incubator	Thermo Fisher Scientific  model Forma series II water-jacketed CO2 incubator	3140
Cell culture medium
Cell line ES-E14
Chelex-100	BioRad	1422842	200–400 dry mesh size
Chloroform	Sigma	366919
Concanavalin A (ConA)-coated magnetic beads	Polysciences	86057-10
Digitonin	Millipore	300410-5MG
DMSO	Sigma	D8418
E. coli tRNA	Sigma	10109541001
Ethanol	Thermo Fisher Scientific	BP2818100	Handle ethanol with care, it is flammable
glucose	Sigma	G7021
Glycerol	Sigma	G5516
HCl	Sigma	H1758
HEPES	Sigma	H3375
Isopropanol	Thermo Fisher Scientific	BP26324	Handle isoproponol with care, it is flammable
KAPA HiFi HotStart Ready Mix	Roche	7958889001
KCl	Sigma	P9541
KOH	sigma	221473	Handle KOH with care, it is corrosive
Low-speed vacuum
Magnet stand	Millipore	LSKMAGS08
Mastercycler Gradient PCR Thermal Cycler	Eppendorf	5331
MgCl2	Sigma	M8266
Micrococcal nuclease  (MNase)	Worthington	LS004798
MinElute PCR purification kit	Qiagen	28006
Mini-beads beater	Mini BeadBeater 24 Disruptor	607
Mini-centrifuge	Eppendorf	5424R
MnCl2	Sigma	244589
Multiplatform shaker	Fisherbrand	02-217-765
Multiplatform shaker incubator	Benchmark	INCU-SHAKER 10L
Multithermal heat block	Eppendorf	Thermomixer C
N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS)	Sigma	T5130	200–400 dry mesh size
NaCl	Sigma	S9888
NaOH	Sigma	S8045	Handle NaOH with care, it is corrosive
NEBNext HiFi 2× PCR master mix	New England Biolabs	M0541
NP-40 Igepal CA-630	Thermo Scientific	J61055.AP
Paraformaldhyde	sigma	158127	Handle paraformaldehyde with care, it is flammable solid hazardous.
pA-Tn5 enzyme	Addgene	121137
Pefabloc	Sigma	11429868001
Phenol-chloroform-isoamyl alcohol	Invitrogen	15593049
Pipette P1000, P200, P20 and P2	Eppendorf	2231300004
PMSF	Sigma	10837091001
Protease inhibitor cocktail tablet	Sigma	S8830
Protease inhibitor cocktail tablet	Sigma	S8830
Protein G-sepharose beads	GE Healthcare	17-0618-02
Protein precipitation solution	Qiagen	848023
Protein precipitation solution	Qiagen	848023	Handle KOH with care, it is corrosive
Protein precipitation solution	Qiagen	848023
Proteinase K	Sigma	311584401
QIAquick PCR purification kit	Qiagen	28106
RNase A	Sigma	10109169001
SDS	Sigma	L3771
Spectrophotometer	Thermo Scientific.	2000/2000c
Spermidine	Sigma	S2626
T4 DNA Pol	New England Biolabs,	M0203
Table top centrifuge	Beckman Coulter	Allegra X-5
Tris	Sigma	11814273001
Triton X-100	Sigma	T8787
yeast extract	Thermo Fisher Scientific	212730