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Analysis of RB action in DNA damage checkpoint response.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2004; Vol. 281, pp. 3-16. - Publication Year :
- 2004
-
Abstract
- Cell cycle checkpoints play a key role in maintaining genome stability by monitoring the order and integrity of cell division events. Checkpoints induced by DNA damage function to limit the propagation of potentially deleterious mutations. The retinoblastoma tumor suppressor (RB) is a critical effector of DNA damage checkpoint function by eliciting G1-phase cell cycle arrest following genotoxic stress. Here, we describe methodologies for evaluation of three facets of RB action in the DNA damage checkpoint response: (1) transcriptional repression of E2F-regulated genes (cyclin A reporter assay); (2) induction of cell cycle arrest (Brd-U incorporation assay); and (3) inhibition of DNA double-strand break accumulation (phosphorylated-histone H2A.X detection). Together, this combination of techniques allows the evaluation of RB action in the coordinated checkpoint response to DNA damage.
- Subjects :
- Animals
Antineoplastic Agents pharmacology
Bromodeoxyuridine
Cells, Cultured
Cisplatin pharmacology
DNA Replication drug effects
DNA-Binding Proteins metabolism
E2F Transcription Factors
Embryo, Mammalian cytology
Embryo, Mammalian metabolism
Fibroblasts cytology
Genes, Reporter
Histones metabolism
Mice
Mice, Knockout
Retinoblastoma Protein deficiency
Transcription Factors metabolism
Cell Cycle
Cell Cycle Proteins
DNA Damage genetics
Fibroblasts metabolism
Genes, cdc physiology
Retinoblastoma Protein physiology
Subjects
Details
- Language :
- English
- ISSN :
- 1064-3745
- Volume :
- 281
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 15220518
- Full Text :
- https://doi.org/10.1385/1-59259-811-0:003