title: Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells.
abstract: In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naïve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002.
authors.affiliation: Biology Department, University of York, Heslington, York, YO10 5YW, UK.
authors.forename: Katherine
authors.initials: K
authors.lastname: Roper
authors.forename: Dawn
authors.initials: D
authors.lastname: Coverley
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System; D004249:DNA Damage; D004261:DNA Replication; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005047:Etoposide; D016193:G1 Phase; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D013194:Staining and Labeling
chemical_list: D002457:Cell Extracts; C468783:H2AX protein, human; D006657:Histones; D005047:Etoposide