Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability

Sannino, Vincenzo; Pezzimenti, Federica; Bertora, Stefania; Costanzo, Vincenzo

doi:10.1016/bs.mie.2017.03.018

Cited by 8 publications

(3 citation statements)

References 17 publications

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“…Only a few in vitro systems are able to reconstitute complex chromatin with physiological nucleosome spacing [reviewed in (74,75)]. The most widely used system for assembly of dynamic chromatin that responds to DNA damage is derived from Xenopus eggs, where repair of pyrimidine dimers, interstrand crosslinks, topoisomerase II adducts and O6-methylguanine have been studied (6,76–78). Xenopus egg extracts have also been used very successfully to study the loading and unloading of proteins to DSBs (21,79–82), NHEJ (83), the replication at DSBs (84) and DSB repair during mitosis (85).…”

Section: Discussionmentioning

confidence: 99%

“…Cell-free systems can be used to mechanistically understand the processes revolving around damaged chromatin. The two most prominent experimental systems for the reconstitution of chromatin with physiological properties are derived from Xenopus laevis eggs or oocytes (6), and preblastoderm embryos of Drosophila melanogaster (7,8). In both models, the fertilized eggs contain large stockpiles of maternal proteins and RNA that support the first 12 cell divisions, or 13 nuclei divisions, respectively, in the absence of significant transcription (9).…”

Section: Introductionmentioning

confidence: 99%

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A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

Harpprecht

Baldi

Schauer

et al. 2019

Nucleic Acids Research

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Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

Harpprecht

Baldi

Schauer

et al. 2019

Nucleic Acids Research

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“…DNA templates. Demembranated sperm nuclei were prepared as previously described 44 . Briefly, X. laevis males were primed with 50 U PMSG (Folligon) 7 days in advance and with 300 U HCG the day before testis dissection.…”

Section: Methodsmentioning

confidence: 99%

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development

et al. 2020

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In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.

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DNA Damage Response in Xenopus laevis Cell-Free Extracts

Aparicio

Gautier

2021

Cell Cycle Checkpoints

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Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability

Cited by 8 publications

References 17 publications

A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

A Drosophila cell-free system that senses DNA breaks and triggers phosphorylation signalling

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development

DNA Damage Response in Xenopus laevis Cell-Free Extracts

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