Dataset Information

REV7 associates with ATRIP and FAM35A

ABSTRACT: REV7 is a remarkably multi-faceted protein with important and separable roles in multiple genome maintenance pathways including Trans-Lesion Synthesis, the Fanconi Anemia pathway, as well as p53 signaling and Double Stranded Break resection. To investigate novel functions and pathways involving REV7, we performed mass-spectrometry analysis and identified proteins associated with REV7. We discovered two novel factors: the ATR-ATRIP complex and FAM35A. The REV7-FAM35A interaction was reported in a previous paper. Here, we identified the ATR-binding partner and activator ATRIP as a new REV7-associated protein. Through a comprehensive series of in vitro biochemical studies and cell-based assays we demonstrate that ATRIP is a direct binding partner of REV7, and that REV7-ATRIP interactions are critical for negative regulation of ATR activity. Our findings establish a new mechanism for negative regulation of ATR by the DNA repair protein REV7.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Junya Tomida

LAB HEAD: Junya Tomida

PROVIDER: PXD072142 | Pride | 2026-01-22

REPOSITORIES: Pride

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REV7 associates with ATRIP and inhibits ATR kinase activity.

Biller Megan M Kabir Sara S Nipper Sarah S Allen Sydney S Kayali Yara Y Kuncik Skyler S Sasanuma Hiroyuki H Zhou Pei P Vaziri Cyrus C Tomida Junya J

Nucleic acids research 20260101 2

Ataxia-telangiectasia mutated and RAD3-related (ATR) and its partner ATR-interacting protein (ATRIP) function as a critical proximal sensor and transducer of the DNA damage response (DDR). Several ATR substrates, including p53 and CHK1, are crucial for the coordination of cell cycle phase transitions, transcription, and DNA repair when cells sustain DNA damage. While much is known about ATR activation mechanisms, it is less clear how ATR signaling is negatively regulated in cells. Here, we ident ...[more]

PMID: 41562258

Publication: 1/3

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Project description:ATR is a PI3K-like kinase protein, regulating checkpoint responses to DNA damage and replication stress. Apart from its checkpoint function in the nucleus, ATR actively engage in antiapoptotic role at mitochondria following DNA damage. The different functions of ATR in the nucleus and cytoplasm are carried out by two prolyl isomeric forms of ATR: trans- and cis-ATR, respectively. The isomerization occurs at the Pin1 Ser428-Pro429 motif of ATR. Here we investigated the structural basis of the subcellular location-specific functions of human ATR. Using a mass spectrometry-based footprinting approach, the surface accessibility of ATR lysine residues to sulfo-NHS-LC-biotin modification was monitored and compared between the cis and the trans-isomers. We have identified two biotin-modified lysine residues, K459 and K469, within the BH3-like domain of cis-ATR that were not accessible in trans-ATR, indicating a conformational change around the BH3 domain between cis- and trans-ATR. The conformational alteration also involved the N-terminal domain and the middle HEAT domain. Moreover, experimental results from an array of complementary assays show that cis-ATR with the accessible BH3 domain was able to bind to tBid while trans-ATR could not. In addition, both cis- and trans-ATR can directly form homodimers via their C-terminal domains without ATRIP, while nuclear (trans-ATR) in the presence of ATRIP forms dimer-dimer complexes involving both N- and C-termini of ATR and ATRIP after UV. Structural characteristics around the Ser428-Pro429 motif and the BH3 domain region are also analyzed by molecular modelling and dynamics simulation. In support, cis conformation was found to be significantly more energetically favourable than trans at Ser428-Pro429 bond in a 20-aa wild-type ATR peptide. Taken together, our results suggest that the isomerization-induced structural changes of ATR define both its subcellular location and compartment-specific functions and plays an essential role in promoting cell survival and DNA damage responses.

Project description:Genotoxic agents can cause replication fork stalling in dividing cells due to DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. The methodology is generic and is widely applicable in the ubiquitin field. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 hours of Hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and 2 proteins were down-regulated for SUMOylation, whereas after 24 hours, 35 proteins were up-regulated for SUMOylation and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1,000 SUMO-2 acceptor lysines in target proteins. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1 and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage

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