Study of the DNA Damage Checkpoint using &lt;em&gt;Xenopus&lt;/em&gt; Egg Extracts

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H 2 O 2 ) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H 2 O 2 -induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H 2 O 2 -damaged chromatin. APE2 3′-phosphodiesterase and 3′-5′ exonuclease activity is essential for single-stranded DNA generation in the 3′-5′ direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response. C ells are constantly challenged by exogenous and endogenous insults that threaten genomic integrity. Excess accumulation of reactive oxygen species leads to oxidative DNA damage, such as DNA strand breaks with 3′-modified termini, which is often the underlying pathology in a variety of diseases including neurodegenerative diseases and cancer (1-6). Cellular responses to DNA damage are mainly coordinated by two distinct DNA damage checkpoint signaling cascades: ATM (ataxia-telangiectasia mutated)-checkpoint kinase 2 (Chk2) and ATR (ATM and Rad3-related)-checkpoint kinase 1 (Chk1) pathways (7-10). ATM is activated by intermolecular autophosphorylation and dimer dissociation in response to double-stranded beaks (DSBs) (11-13). ATR is activated by primed single-stranded DNA (ssDNA) in response to a variety of DNA damage or replication stresses (14,15). Oxidative stress has been demonstrated to activate an ATMdependent DNA damage checkpoint (16-18). However, in previous studies, hyperoxic conditions resulted in the phosphorylation of Chk1 and p53 in an ATR-dependent but ATM-independent fashion (19). Furthermore, it remains unclear which specific DNA structures trigger checkpoint signaling during oxidative stress.To eliminate oxidative DNA damage, base excision repair (BER) has evolved as a major DNA damage repair mechanism (20). In the initial step of BER, o...

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“…Xenopus egg extracts were prepared as described previously (28,30). Sperm chromatin was obtained through previously described methods (71).…”

Section: Methodsmentioning

confidence: 99%

“…We performed a series of studies in checkpoint signaling using Xenopus egg extracts, a well-characterized cell-free model system (28)(29)(30)(31)(32). Here, we provide evidence that suggests APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress.…”

mentioning

confidence: 96%

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

Willis

Patel

Lentz

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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149

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“…The care and use of X. laevis were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Charlotte. X. laevis egg extracts were prepared as described previously (55,56). The X. laevis egg extracts and related experiments with recombinant proteins and antibodies are described in SI Appendix, Materials and Methods.…”

Section: Ape2 Protein Expression the Ape2mentioning

confidence: 99%

APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

Wallace

Berman

Mueller

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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104

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The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3′-5′ nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structure and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3′-5′ exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.oxidative stress | APE2 | Zf-GRF | crystallography | Xenopus laevis

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“…The preparation of Xenopus egg extract for β-catenin degradation (intermediate speed extract) differs from classic extracts prepared for studying cell cycle 30–32 , which are low-speed or high-speed extracts 35 . Adapting Xenopus egg extract for optimal degradation of other proteins may require altering the centrifugation speed and number of spins.…”

Section: Discussionmentioning

confidence: 99%

“…Methods for the preparation of Xenopus egg extract for studying the cell cycle have been described in previous JoVE publications 30–32 . The current protocol describes a modification of these methods and is optimized for the degradation of [ 35 S]-radiolabeled β-catenin and luciferase-tagged β-catenin in Xenopus egg extract.…”

Section: Introductionmentioning

confidence: 99%

Reconstitution Of β-catenin Degradation In <em>Xenopus</em> Egg Extract

Chen

Broadus

Huppert

et al. 2014

JoVE

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Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1–3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, β-catenin. Two different methods are described to assess β-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess β-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of β-catenin degradation.

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Study of the DNA Damage Checkpoint using <em>Xenopus</em> Egg Extracts

Cited by 31 publications

References 14 publications

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

Reconstitution Of β-catenin Degradation In <em>Xenopus</em> Egg Extract

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Study of the DNA Damage Checkpoint using <em>Xenopus</em> Egg Extracts

Cited by 31 publications

References 14 publications

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress

APE2 Zf-GRF facilitates 3′-5′ resection of DNA damage following oxidative stress

Reconstitution Of &#946;-catenin Degradation In <em>Xenopus</em> Egg Extract

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Reconstitution Of β-catenin Degradation In <em>Xenopus</em> Egg Extract