Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.
Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.
Lupardus
Patrick J
PJ
Department of Molecular Pharmacology, Stanford University, 318 Campus Drive , Stanford, CA 94305-5441, USA.
Van
Christopher
C
Cimprich
Karlene A
KA
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000502:physiology; D002457:Cell Extracts / Q000737:chemistry* / Q000235:genetics; D004261:DNA Replication / Q000502:physiology; D004587:Electrophoresis, Agar Gel; D010063:Ovum / Q000737:chemistry*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry* / Q000502:physiology
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D029867:Xenopus Proteins; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases