Methods for Studying DNA Single-Strand Break Repair and Signaling in Xenopus laevis Egg Extracts

Abstract

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.

APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells

“…The preparation of Xenopus HSS and the similar setup of SSB-induced DDR pathway assays for Figure 3B were recently described ( Willis et al, 2012 ; Yan and Willis, 2013 ; DeStephanis et al, 2015 ; Cupello et al, 2016 ; Lin et al, 2018 , 2019 ). Briefly, SSB plasmid or control plasmid was added to the HSS to a final concentration of 75 ng/μL and incubated for 30 min at room temperature.…”

“…After transformation into DH5alpha E. coli, plasmids were prepared via QIAprep Spin Miniprep Kit (QIAGEN Cat#27106) following vendor's protocol. In addition, the control (CTL) plasmid, SSB plasmid, FAM-labeled 70-nt ssDNA, and FAM-labeled 70-bp dsDNA with a gap or nicked structure in Figure 3 were described previously (Lin et al, 2018(Lin et al, , 2019(Lin et al, , 2020. The pET32a-hAPE2 was described previously (Tsuchimoto et al, 2001).…”

Involvement of SAMHD1 in dNTP homeostasis and the maintenance of genomic integrity and oncotherapy (Review)

“…DNA damage in cells, mainly single-strand breaks, arises frequently (approximately 10,000 lesions per cell per day) by a variety of endogenous and exogenous stimuli (61,62). It has been well established that the DNA damage response (DDR) pathway detects lesions in DNA strands and activates the repair system (63).…”

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“…The preparation of Xenopus HSS and the similar setup of SSB-induced DDR pathway assays for Figure 3B were recently described (Willis et al, 2012;Yan and Willis, 2013;DeStephanis et al, 2015;Cupello et al, 2016;Lin et al, 2018Lin et al, , 2019. Briefly, SSB plasmid or control plasmid was added to the HSS to a final concentration of 75 ng/µL and incubated for 30 min at room temperature.…”

“…Dihydrocelastryl is structurally similar to Celastrol, which is a natural compound derived from thunder god vine and has been implicated in therapies for cancers such as pancreatic cancer and prostate cancer as an HSP90 modulator and/or proteasome inhibitor (Figure 3A; Hieronymus et al, 2006;Zhang et al, 2008). We recently characterized the requirement of APE2 in the ATR-Chk1 DDR pathway activation in response to defined SSB structures in the Xenopus high-speed supernatant (HSS) system (Willis et al, 2012;Cupello et al, 2016;Lin et al, 2018Lin et al, , 2019. First, we intended to test whether Celastrol affects the SSB-induced ATR-Chk1 DDR pathway.…”