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DNA single-strand breaks (SSBs) are the most common type of DNA lesions as they are generated approximately 10,000 times per mammalian cell each day. Unrepaired SSBs compromise DNA replication and transcription programs, leading to genome instability, and have been implicated in many diseases including cancer. In this chapter, we introduce methods to study the ATR-Chk1 DNA damage response (DDR) pathway and DNA repair pathway in response to a site-specific, defined SSB plasmid in Xenopus laevis egg extracts. This experimental system can be applied in future studies to reveal many aspects of the molecular mechanisms of SSB repair and signaling in eukaryotes.
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