The RPA-binding domain and the KKRK motif in Rad26ATRIP cooperate at the perturbed DNA replication fork for initiating checkpoint signaling
Yong-jie Xu, Anmin Gao, Kamal Dev, Yuyuan Zheng, Mashael Y. Alyahya, Sairam Pasam, Guramrit Kaur, Chun Zhou
Abstract
Rad26 is the homolog of human ATRIP and budding yeast Ddc2 in Schizosaccharomyces pombe. Like ATRIP and Ddc2, Rad26 works with Rad3ATR/Mec1 to initiate checkpoint signalling in response to perturbed DNA replication and various types of DNA damage. To better understand the checkpoint initiation mechanism in fission yeast, we carried out genetic and biochemical analyses on the N-terminus of Rad26. Although Rad26 homologs do not share much sequence similarity, we demonstrate that, like ATRIP and Ddc2, Rad26 possesses a replication protein A (RPA) binding domain (RBD) in its N-terminus, suggesting a highly conserved mechanism.
Introduction
Genome integrity is constantly challenged by numerous cellular and environmental factors that damage DNA or disrupt the accurate transmission of heritable information to the next generation [1,2]. DNA damage is also a cornerstone of cancer therapy. To maintain genome integrity, eukaryotes have evolved several dedicated mechanisms, including high-fidelity DNA replication, repair pathways of various types of DNA damage and replication errors, and the cell-cycle checkpoint system.
Materials and method
Yeast strains and plasmids. The S. pombe strains were cultured at 30°C in YE6S (0.5% yeast extract, 3% dextrose, and 6 supplements) or synthetic EMM6S medium lacking the appropriate supplement [54]. Yeast strains, plasmids, and PCR primers used in this study are listed in Tables A, B, and C in S1 File, respectively.
Results
The N-terminal region of Rad26ATRIP promotes Rad3ATR kinase signalling. The fission yeast Rad26, like human ATRIP and budding yeast Ddc2, binds to Rad3ATR and promotes Rad3 kinase signalling in both the DRC and the DDC pathways [16,22]. In the DRC pathway, Rad3-Rad26 activates the effector kinase Cds1CHK2 at the perturbed fork via the mediator protein Mrc1Claspin [23].
Discussion
To better understand the checkpoint initiation function of Rad26ATRIP, we used an integrated approach of genetic, biochemical, and structural modelling, focusing on the N-terminal region. Our in vivo and in vitro studies identified an RBD in the Rad26 N-terminus and the key residues in the RBD that bind to the F-domain of Ssb1, the large subunit of RPA in S. pombe.
Acknowledgments
We thank NBRP/YGRC in Japan for the yeast strains. We also thank other members of the Xu lab and the Zhou lab for their help and critical reading of the manuscript.
Citation: Xu Y-j, Gao A, Dev K, Zheng Y, Alyahya MY, Pasam S, et al. (2026) The RPA-binding domain and the KKRK motif in Rad26ATRIP cooperate at the perturbed DNA replication fork for initiating checkpoint signalling. PLoS Genet 22(2): e1012052. https://doi.org/10.1371/journal.pgen.1012052
Editor: Michael Snyder, Stanford University School of Medicine, UNITED STATES OF AMERICA
Received: September 28, 2025; Accepted: February 9, 2026; Published: February 23, 2026
Copyright: © 2026 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are in the manuscript and its Supporting information files.
Funding: This study was supported by NIH/National Institute of General Medical Sciences, grant R35GM144307 to YjX and the National Natural Science Foundation of China, grant 32371250 to CZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
