Исследование Checkpoint повреждения ДНК использовании<em> Xenopus</em> Экстракты Egg

Summary

Xenopus яйцо экстракт является полезной модели системы для расследования контрольно-пропускного пункта повреждения ДНК. Этот протокол предназначен для подготовки Xenopus яйцо экстракты и повреждение ДНК реагенты контрольно-пропускном пункте вызывающим. Эти методы могут быть адаптированы к различным повреждающим ДНК подходы в изучении контрольно-пропускного пункта сигнализации повреждения ДНК.

Abstract

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.

Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group¹. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.

The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress ². Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures ³. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin^{3, 4}. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint ^4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint ⁸. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy’s laboratory and is widely used to induce ATR/Chk1 checkpoint signaling ^9-12.

Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Protocol

1. Подготовка LSE Женский лягушек (Xenopus Хепориз) вводится два раза для сбора яиц. Первая инъекция (грунтовка) составляет 100 U PMSG (Беременные Mare сыворотке гонадотропина) в лягушку. Лягушки должны быть загрунтованы по крайней мере, за два дня до вызывающие откладки яиц и загрунтоват?…

Discussion

Есть несколько преимуществ в изучении контрольно-пропускного пункта повреждения ДНК использовании Xenopus яйцо экстрактов. Использование яйцо экстракт содержит большое количество бесклеточных экстрактов синхронизированы в интерфазе клеточного цикла. Яйцо экстракты могут быть ле…

Materials

			Reagents
Anti-Chk1 P-S344 antibody	Cell Signaling	2348L
Anti-Chk1 antibody	Santa Cruz	SC7898
Aprotinin	MP Biomedicals	0219115880
Cycloheximide	Sigma	C7698-5G
Cytochalasin B	EMD	250233
Dithiothreitol (DTT)	VWR	JTF780-2
hCG	Sigma	CG10-10VL
L-Cysteine	Sigma	C7352-1KG
Leupeptin	VWR	97063-922
Methyl methanesulfonate (MMS)	Sigma	129925-5G
Nocodazole	Sigma	M1404-2MG
PMSG	Calbiochem	367222
Sample buffer	Sigma	S3401
Tautomycin	Wako Chemicals USA	209-12041
			Equipment
Bucket for egg laying	Rubbermaid commercial products	6308
CL2 IEC centrifuge with swinging bucket rotor	Thermo Scientific	004260F
HB6 swinging bucket rotor	Thermo Scientific	11860
Sorvall RC6 plus superspeed centrifuge	Thermo Scientific	46910
UV crosslinker	UVP	95-0174-01
			Solutions
1x MMR			100 mM NaCl, 2 mM KCl, 0.5 mM MgSO4, 2.5 mM CaCl2, 5 mM HEPES, adjust pH to 7.8 with 10 M NaOH
Aprotinin/Leupeptin stock			10 mg/ml each in water. Store 20 μl aliquots at -80 °C.
Buffer X			0.2 M sucrose, 80 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, adjust pH to 7.5 by HCl
Cycloheximide stock			10 mg/ml in water. Store 1 ml aliquots at -20 °C.
Cytochalasin B stock			5 mg/ml in DMSO. Store 20 μl aliquots at -20 °C.
Dithiothreitol (DTT) stock			1 M in water. Store 1 ml aliquots at -20 °C.
ELB			0.25 M sucrose, 1 mM DTT, 50 μg/ml cycloheximide, 2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES, pH7.7
Nocodazole stock			10 mg/ml in DMSO. Store 5 μl aliquots at -80 °C.
Energy Mixture			375 mM creatine phosphate, 50 mM ATP, and 25 mM MgCl2. Aliquots are saved at -80 °C.
Nuclear dye solution			0.4 μg/ml Hoechst 33258, 25% glycerol (v/v), in 1x PBS
Tautomycin stock			100 μM in DMSO. Store 10 μl aliquots at -80 °C.