Abstract:
:Although many players of the DNA damage response and DNA repair have been identified in several systems their biochemical role is still poorly understood. The use of the Xenopus laevis egg extract cell-free system allowed biochemical dissection of DNA replication and cell cycle events in a complex biological context. The possibility of manipulating the protein content by using protein depletion procedures makes egg extract a powerful system to study proteins whose inactivation results in cellular lethality. The egg extract has been increasingly used to study DNA damage response and the coordination of DNA replication with DNA repair. The recent development of advanced imaging techniques based on electron microscopy has allowed the characterization of replication intermediates formed in the absence of essential DNA repair proteins. These studies have been important to understand how cells maintain genome stability under unchallenged and stressful conditions. Here, we present a collection of protocols that have been developed to recapitulate DNA damage response activated by chromosome breakage in egg extract and to isolate replication intermediates for electron microscopy analysis using sperm nuclei or more defined genomic substrates.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Sannino V,Pezzimenti F,Bertora S,Costanzo Vdoi
10.1016/bs.mie.2017.03.018subject
Has Abstractpub_date
2017-01-01 00:00:00pages
211-232eissn
0076-6879issn
1557-7988pii
S0076-6879(17)30113-1journal_volume
591pub_type
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