{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T22:14:19Z","timestamp":1793398459311,"version":"4.5.0"},"posted":{"date-parts":[[2026,2,9]]},"group-title":"Biochemistry","reference-count":84,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T00:00:00Z","timestamp":1770595200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["ES023350"],"award-info":[{"award-number":["ES023350"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,13]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Oxidative DNA damage caused by endogenous reactive oxygen species (ROS) is a key driver of mutagenesis, cellular dysfunction, and aging, contributing to diseases like cancer, neurodegeneration, rheumatoid arthritis, cardiovascular disorders, and diabetes. Although more than 20 oxidative base lesions have been identified, ROS-induced DNA-protein crosslinks (DPCs) are poorly characterized. ROS-DPCs are unusually bulky and highly toxic lesions that accumulate in metabolically active tissues with age, but their identities, biological consequences, and repair in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H\n                  <jats:sub>2<\/jats:sub>\n                  O\n                  <jats:sub>2<\/jats:sub>\n                  ) and elucidated the mechanisms of their removal. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. Our data further reveal that DNA replication and transcription facilitate DPC detection and identify a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog (\u03a8-GSH), suggesting a possible therapeutic strategy for preventing diseases associated with increased ROS levels.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>KEY POINTS<\/jats:title>\n                  <jats:p>Mass spectrometry-based proteomics identified over 100 proteins participating in DNA-protein cross-links in human cells treated with ROS<\/jats:p>\n                  <jats:p>Our work reveals the mechanisms through which living cells recognize and remove ROS-DPCs<\/jats:p>\n                  <jats:p>Our study demonstrates the potential of a glutathione analog to prevent ROS-DPC formation<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>GRAPHICAL ABSTRACT<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"704426v2_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.02.06.704426","type":"posted-content","created":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T01:40:38Z","timestamp":1770687638000},"source":"Crossref","is-referenced-by-count":1,"title":["Living Cells Employ Ubiquitin-Proteasomal System and Nucleotide Excision Repair Pathways to Remove Reactive Oxygen Species-Induced DNA-Protein Crosslinks (ROS-DPCs)"],"prefix":"10.64898","author":[{"given":"Cesar I","family":"Cyuzuzo","sequence":"first","affiliation":[{"name":"Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Department of Pharmacology, University of Minnesota, Minneapolis, MN. 55455, 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