Dataset Information

Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

ABSTRACT: The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1's nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

SUBMITTER: Montales K

PROVIDER: S-EPMC7801695 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

Montales Katrina K Kim Ahhyun A Ruis Kenna K Michael W Matthew WM

Scientific reports 20210111 1

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DS ...[more]

PMID: 33432091

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Project description:Cell-free Xenopus egg extract is a widely used and biochemically tractable model system that allows recapitulation and elucidation of fundamental cellular processes. Recently, the introduction of microfluidic extract manipulation has enabled compartmentalization of bulk extract and a newfound ability to study organelles on length scales that recapitulate key features of cellular morphology. While the microfluidic confinement of extracts has produced a compelling platform for the in vitro study of cell processes at physiologically-relevant length scales, it also imposes experimental limitations by restricting dynamic control over extract properties. Here, we introduce photodegradable polyethylene glycol (PEG) hydrogels as a vehicle to passively and selectively manipulate extract composition through the release of proteins encapsulated within the hydrogel matrix. Photopatterned PEG hydrogels, passive to both extract and encapsulated proteins, serve as protein depots within microfluidic channels, which are subsequently flooded with extract. Illumination by ultraviolet light (UV) degrades the hydrogel structures and releases encapsulated protein. We show that an engineered fluorescent protein with a nuclear localization signal (GST-GFP-NLS) retains its ability to localize within nearby nuclei following UV-induced release from hydrogel structures. When diffusion is considered, the kinetics of nuclear accumulation are similar to those in experiments utilizing conventional, bulk fluid handling. Similarly, the release of recombinant cyclin B ?90, a mutant form of the master cell cycle regulator cyclin B which lacks the canonical destruction box, was able to induce the expected cell cycle transition from interphase to mitosis. This transition was confirmed by the observation of nuclear envelope breakdown (NEBD), a phenomenological hallmark of mitosis, and the induction of mitosis-specific biochemical markers. This approach to extract manipulation presents a versatile and customizable route to regulating the spatial and temporal dynamics of cellular events in microfluidically confined cell-free extracts.

Dataset Information

Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

Publications

Structure-function analysis of TOPBP1's role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system.

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