title: Study of the DNA damage checkpoint using Xenopus egg extracts.
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 group. 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. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures. 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. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint. 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. 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.
authors.affiliation: Department of Biology, University of North Carolina at Charlotte, USA.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Willis
authors.forename: Darla
authors.initials: D
authors.lastname: DeStephanis
authors.forename: Yogin
authors.initials: Y
authors.lastname: Patel
authors.forename: Vrushab
authors.initials: V
authors.lastname: Gowda
authors.forename: Shan
authors.initials: S
authors.lastname: Yan
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D005260:Female; D008297:Male; D016685:Mitomycin / Q000494:pharmacology; D010063:Ovum / Q000737:chemistry*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D013094:Spermatozoa / Q000737:chemistry; D014020:Tissue Extracts / Q000737:chemistry* / Q000302:isolation & purification; D014466:Ultraviolet Rays; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D014020:Tissue Extracts; D029867:Xenopus Proteins; D016685:Mitomycin; D004247:DNA; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases