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Chromatinized DNA is under constant assault from endogenous and exogenous sources of damage, which must be effectively repaired to preserve genome stability. Single-strand breaks (SSBs) with chemically heterogeneous DNA ends are one of the most prevalent forms of genomic DNA damage. These SSBs must be enzymatically processed prior to downstream gap-filling DNA synthesis and\/or nick ligation during single-strand break repair (SSBR). Polynucleotide kinase phosphatase (PNKP) is a multifunctional end-processing enzyme that possesses two catalytic activities important for converting non-ligatable SSBs into ligatable SSBs. To date, a mechanistic description for how PNKP processes non-ligatable SSBs in the context of chromatin to initiate SSBR remains undefined. Here, we utilize a combination of biochemical assays and cryogenic electron microscopy (cryo-EM) to define the structural basis of end processing in the nucleosome by PNKP. Cryo-EM structures of PNKP engaged with non-ligatable SSBs at three unique positions within the nucleosome reveal that PNKP locally deforms nucleosomal DNA to reposition the SSBs into the kinase and phosphatase active sites, providing a structural basis for the efficient processing of SSBs throughout the nucleosome. Additional cryo-EM structures reveal the PNKP FHA domain also engages the nucleosome acidic patch during non-ligatable SSB recognition, which accelerates the processing of non-ligatable SSBs in the nucleosome. Together, these findings provide important mechanistic insight into the initial end processing step of chromatin-based SSBR.<\/jats:p>","DOI":"10.64898\/2026.08.12.743989","type":"posted-content","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T04:50:23Z","timestamp":1786683023000},"source":"Crossref","is-referenced-by-count":0,"title":["Structural basis of end processing in the nucleosome by polynucleotide kinase phosphatase"],"prefix":"10.64898","author":[{"given":"Daniel J.","family":"Boesch","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA"},{"name":"University of Virginia Comprehensive Cancer Center, Charlottesville, Virginia, 22908, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nadia I.","family":"Martin","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John J.","family":"Evans","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tyler M.","family":"Weaver","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, 22908, USA"},{"name":"University of Virginia Comprehensive Cancer Center, Charlottesville, Virginia, 22908, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081707200483000_2026.08.12.743989v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/38444"},{"key":"2026081707200483000_2026.08.12.743989v1.2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.225771.117"},{"key":"2026081707200483000_2026.08.12.743989v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.10.004"},{"key":"2026081707200483000_2026.08.12.743989v1.4","doi-asserted-by":"crossref","unstructured":"Cordero, C. et al. 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