Xenopus Cell-Free Extracts to Study DNA Damage Checkpoints

Surveillance mechanisms monitoring genomic integrity operate through signal transduction pathways called checkpoints. Checkpoints are essential to delay cell cycle progression in response to deoxyribonucleic acid (DNA) damage (1 ,2 ). These pathways require the coordinated monitoring and sensing of the damaged DNA with downstream signaling ultimately leading to cell cycle arrest. Traditionally, the DNA damage response and its checkpoint’s component have been studied mostly using yeast and mammalian cells. Budding and fission yeasts have been instrumental to identify mutations in genes impaired in DNA damage cell cycle checkpoints. Genetic screens in yeast have been a successful approach to identify radiation-sensitive (rad mutants) and checkpoint genes (hus, mad, bub mutants) participating in the maintenance of genomic integrity (3 ). However, this approach has several limitations. Essential genes may never be isolated in standard genetic screens. DNA damage response is more complex in vertebrates than it is in yeast and critical regulators of the DNA damage response, such as p53 and BRCA1 , are found only in vertebrates (2 ). Mammalian cell lines from diverse origins, including some derived from patients harboring defects in the DNA damage response, have also been used extensively to study the DNA damage response (4 –6 ). However, these cell-based systems do not allow using specific biochemical readouts as they are based on phenotypes resulting from complex outputs such as cell growth or survival.