Cell-free Xenopus egg extracts for studying DNA damage response pathways

Abstract

In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol κ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts.

APE2 promotes DNA damage response pathway from a single-strand break

“…Sperm chromatin was prepared and utilized according to methods as described previously ( 11 , 24 , 35 , 39 ). The preparation of Xenopus LSS, HSS and NPE were described previously ( 35 , 36 , 39 ). DNA synthesis analysis from the HSS/NPE system was performed as previously described ( 11 , 35 ).…”

“…As a cell-free experimental system from eggs of the African clawed frog, Xenopus egg extracts have been widely used in studies of chromosome metabolism, and findings from Xenopus system can be validated in mammalian cell lines ( 33–35 ). Three different types of Xenopus egg extracts have been commonly used: low-speed supernatant (LSS), high-speed supernatant (HSS), and nucleoplasmic extracts (NPE) ( 36–38 ). Here, we have developed a plasmid-based SSB structure at a defined location and found that an ATR-dependent but replication-independent DDR pathway is activated by the defined SSB structure in the Xenopus HSS system.…”

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

“…In particular, mechanistic studies using Xenopus system have provided critical insights into the molecular details of DNA repair and DDR pathways in response to DNA double-strand breaks (DSBs) and inter-strand crosslinks (ICLs) [15–19]. There are several different types of Xenopus egg extracts: low-speed supernatant (LSS), high-speed supernatant (HSS), and nucleoplasmic extracts (NPE) [20,21]. The detailed approaches to prepare these Xenopus egg extracts have been described previously [7,20,22].…”

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

“…Studies in Xenopus have already had major contributions to cancer research [for a comprehensive overview see (Hardwick and Philpott, )]. First of all, Xenopus research yielded important knowledge on processes deregulated in cancers such as cell cycle regulation, DNA replication and DNA damage responses, which can be very conveniently studied in Xenopus cell‐free oocyte extracts (Cupello et al ., ; Philpott and Yew, ). Next to this, substantial Xenopus research has focused on developmental signaling pathways such as Wnt, Shh (sonic hedgehog), Notch, TGF‐β (transforming growth factor β), and BMP (bone morphogenetic protein) (Cornell and Eisen, ; Fellgett et al ., ; Gradl et al ., ; Grieco and Hlusko, ; Ishizuya‐Oka and Hasebe, ; Montagner et al ., ; Niehrs, ; Sokol, ; Wang and Steinbeisser, ), which are often hijacked by cancer cells to sustain abnormal growth and to promote cancer progression (Aiello and Stanger, ; Bailey et al ., ).…”