Dataset Information

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress

ABSTRACT: O-linked N-acetylglucosamine (O-GlcNAc) is the only mono-saccharide modification occurring in the cytoplasm, nucleus and mitochondria. Recent advent of the mass spectrometry technology has enabled identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, Etoposide, diminishes the association. Using stepped collision energy higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon Etoposide treatment, and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress

ORGANISM(S): Homo Sapiens

SUBMITTER: Jing Li

PROVIDER: PXD053321 | iProX | Fri Jun 21 00:00:00 BST 2024

REPOSITORIES: iProX

ACCESS DATA

Publications

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress.

Zhao Jianxin J Shao Guangcan G Lu Xiaoxuan X Lv Zhuan Z Dong Meng-Qiu MQ Liu Xiaoqian X Li Jing J

The Journal of biological chemistry 20241102 12

O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus, and mitochondria. The recent advent of mass spectrometry technology has enabled the identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication, and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivo ...[more]

PMID: 39491647

Similar Datasets

Project description:Drosophila development is a complex and dynamic process regulated, in part, by members of the Polycomb (Pc), Trithorax (Trx) and Compass chromatin modifier complexes. O-GlcNAc Transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers N-acetyl-D-glucosamine (GlcNAc) onto hydroxyl groups of serine or threonine residues of key transcriptional regulators using the nutrient-derived UDP-GlcNAc as a substrate, which is dynamically removed by O-GlcNAcase (OGA). We performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells and found that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. To examine the in vivo consequences of disturbed O-GlcNAc cycling in the whole animal, we produced a null allele of oga (ogadel.1) in Drosophila. Epigenetic activators including Trx group members Trithorax (Trx), Absent small or homeotic discs 1 (Ash1) and Compass member Set1 histone methyltransferases are O-GlcNAc modified in ogadel.1 mutants. ogadel.1 mutants displayed altered expression of a distinct set of cell cycle related genes in ovaries. Our results suggest that the loss of OGA could affect epigenetic machinery by accumulating O-GlcNAc on numerous chromatin factors including Trx, Ash1 and Set1 in Drosophila. We performed affymetrix tilingarray analysis after OGT or OGA RNAi knockdown in Drosophila S2 cells to find if that O-GlcNAc was elevated genome wide particularly at genes related to mitosis and cell cycle in OGA RNAi cells, but not at sites co-occupied by Pc member Pleiohomeotic (Pho), ------------------------------- This represents the gene expression component only

Dataset Information

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress

Publications

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets