Xenopus cell-free extracts to study the DNA damage response.
Cell-free systems derived from Xenopus eggs represent a powerful tool, intermediate between in vitro and in vivo model systems. Here, we describe protocols to prepare cell-free extracts recapitulating several aspects of the DNA damage response, including the DNA damage-dependent activation of ATM/ATR protein kinases and several DNA damage checkpoint signaling pathways that inhibit initiation of DNA replication. We provide protocols to prepare cell-free extracts, DNA templates, protein kinase substrates, and to perform checkpoint assays. In addition, we describe related methods that provide useful readouts of the DNA damage response.
Costanzo
Vincenzo
V
Department of Genetics and Development, Columbia University, New York, NY, USA.
Robertson
Kirsten
K
Gautier
Jean
J
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D005260:Female; D005821:Genetic Techniques; D006657:Histones / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D010766:Phosphorylation; D015398:Signal Transduction; D014982:Xenopus laevis
D018797:Cell Cycle Proteins; D002843:Chromatin; C468783:H2AX protein, human; D006657:Histones; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine