{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,9]],"date-time":"2026-08-09T21:22:59Z","timestamp":1786310579695,"version":"build-2736575974"},"posted":{"date-parts":[[2025,7,12]]},"group-title":"Molecular Biology","reference-count":97,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,12]],"date-time":"2025-07-12T00:00:00Z","timestamp":1752278400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01CA291546"],"award-info":[{"award-number":["R01CA291546"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01CA225637"],"award-info":[{"award-number":["R01CA225637"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R03CA270663"],"award-info":[{"award-number":["R03CA270663"]}],"id":[{"id":"https:\/\/ror.org\/040gcmg81","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000066","name":"National Institute of Environmental Health Sciences","doi-asserted-by":"crossref","award":["R21ES032966"],"award-info":[{"award-number":["R21ES032966"]}],"id":[{"id":"10.13039\/100000066","id-type":"DOI","asserted-by":"crossref"}]},{"award":["Graduate School Summer Fellowship program"],"award-info":[{"award-number":["Graduate School Summer Fellowship program"]}],"id":[{"id":"https:\/\/ror.org\/04dawnj30","id-type":"ROR","asserted-by":"publisher"}]},{"award":["OUR Scholars program"],"award-info":[{"award-number":["OUR Scholars program"]}],"id":[{"id":"https:\/\/ror.org\/04dawnj30","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,12]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Poly-ADP-Ribose Polymerase 1 (PARP1) is a potent regulator of DNA damage response signaling through the recruitment of DNA damage repair proteins to damage sites, and its catalytic function of converting Nicotinamide adenine dinucleotide (NAD\n                  <jats:sup>+<\/jats:sup>\n                  ) into poly-ADP-ribose (PAR) which covalently modifies hundreds of protein substrates in a process known as PARylation. However, PARP1\u2019s role in the recognition, processing, and intracellular signaling downstream of DNA damage in cells remains incompletely understood, especially in a replication-independent context. Here, we show that cells exposed to high doses of the methylating agent Methyl Methanesulfonate (MMS) generate DNA double-strand breaks (DSBs) in a base excision repair (BER)-dependent and DNA replication-independent manner. The capacity of cells to generate DSBs after MMS exposure relies heavily on intracellular NAD\n                  <jats:sup>+<\/jats:sup>\n                  availability and PARP1\u2019s catalytic production of PAR. In our experimental system, we show that acute MMS exposure causes NAD\n                  <jats:sup>+<\/jats:sup>\n                  exhaustion in a PARP1-dependent manner, which results in a temporal-dependent loss of downstream PARP1 activity. This functional loss of PARP1 signaling in later timepoints leads to the loss of BER-dependent single-strand break (SSB)-to-DSB conversion, as well as silencing of ATR-Chk1 signaling in both cycling and non-cycling cells, demonstrating a novel PARP1-dependent regulatory mechanism for both ATR-Chk1 signaling and BER-associated processes following methylation challenge. Additionally, we provide experimental evidence supporting the role of PARP1 and NAD\n                  <jats:sup>+<\/jats:sup>\n                  in promoting the exonuclease-mediated SSB-to-DSB conversion. These findings support a previously uncharacterized mechanism of PARP1-mediated replication-independent DSB generation and provide insight into checkpoint signaling by integrating DDR with PARP1\u2019s consumption of NAD\n                  <jats:sup>+<\/jats:sup>\n                  and production of PAR.\n                <\/jats:p>","DOI":"10.1101\/2025.07.10.663928","type":"posted-content","created":{"date-parts":[[2025,7,13]],"date-time":"2025-07-13T05:20:14Z","timestamp":1752384014000},"source":"Crossref","is-referenced-by-count":1,"title":["PARP1 promotes replication-independent DNA double-strand break formation after acute DNA-methylation damage"],"prefix":"10.64898","author":[{"given":"Anne Marie","family":"McMahon","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haichao","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Garrett","family":"Driscoll","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Matos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly","family":"McGhee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jade","family":"Lyttle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8106-6295","authenticated-orcid":false,"given":"Shan","family":"Yan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025071508501003000_2025.07.10.663928v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/0027-5107(90)90173-2"},{"key":"2025071508501003000_2025.07.10.663928v1.2","doi-asserted-by":"publisher","DOI":"10.1093\/carcin\/1.7.595"},{"key":"2025071508501003000_2025.07.10.663928v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrfmmm.2007.05.007"},{"key":"2025071508501003000_2025.07.10.663928v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2010.07.005"},{"key":"2025071508501003000_2025.07.10.663928v1.5","doi-asserted-by":"publisher","DOI":"10.1021\/tx049965c"},{"key":"2025071508501003000_2025.07.10.663928v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/85798"},{"key":"2025071508501003000_2025.07.10.663928v1.7","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M306592200"},{"key":"2025071508501003000_2025.07.10.663928v1.8","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki168"},{"key":"2025071508501003000_2025.07.10.663928v1.9","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201312078"},{"key":"2025071508501003000_2025.07.10.663928v1.10","doi-asserted-by":"publisher","DOI":"10.1007\/BF00267420"},{"key":"2025071508501003000_2025.07.10.663928v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/0027-5107(77)90126-9"},{"key":"2025071508501003000_2025.07.10.663928v1.12","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki681"},{"key":"2025071508501003000_2025.07.10.663928v1.13","doi-asserted-by":"publisher","DOI":"10.1016\/0165-7992(95)00031-3"},{"key":"2025071508501003000_2025.07.10.663928v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrfmmm.2003.12.010"},{"key":"2025071508501003000_2025.07.10.663928v1.15","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.11.7.2221"},{"key":"2025071508501003000_2025.07.10.663928v1.16","doi-asserted-by":"publisher","DOI":"10.1002\/j.1460-2075.1996.tb00890.x"},{"key":"2025071508501003000_2025.07.10.663928v1.17","doi-asserted-by":"publisher","DOI":"10.1021\/tx060164e"},{"key":"2025071508501003000_2025.07.10.663928v1.18","doi-asserted-by":"publisher","DOI":"10.1007\/s002940050157"},{"key":"2025071508501003000_2025.07.10.663928v1.19","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/146.3.797"},{"key":"2025071508501003000_2025.07.10.663928v1.20","doi-asserted-by":"publisher","DOI":"10.1016\/b978-1-4832-3112-9.50015-3"},{"key":"2025071508501003000_2025.07.10.663928v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/35087607"},{"key":"2025071508501003000_2025.07.10.663928v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/27007"},{"key":"2025071508501003000_2025.07.10.663928v1.23","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2008.06.014"},{"key":"2025071508501003000_2025.07.10.663928v1.24","doi-asserted-by":"publisher","DOI":"10.1016\/0027-5107(73)90036-5"},{"key":"2025071508501003000_2025.07.10.663928v1.25","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1006958"},{"key":"2025071508501003000_2025.07.10.663928v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.53"},{"key":"2025071508501003000_2025.07.10.663928v1.27","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2024.01.015"},{"key":"2025071508501003000_2025.07.10.663928v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/j.tcb.2020.02.002"},{"key":"2025071508501003000_2025.07.10.663928v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/s44319-024-00285-5"},{"key":"2025071508501003000_2025.07.10.663928v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2023.12.019"},{"key":"2025071508501003000_2025.07.10.663928v1.31","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkq387"},{"key":"2025071508501003000_2025.07.10.663928v1.32","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl840"},{"key":"2025071508501003000_2025.07.10.663928v1.33","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-32166-7"},{"key":"2025071508501003000_2025.07.10.663928v1.34","doi-asserted-by":"publisher","DOI":"10.1016\/S0301-472X(03)00083-3"},{"key":"2025071508501003000_2025.07.10.663928v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2011.03.004"},{"key":"2025071508501003000_2025.07.10.663928v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.arr.2017.10.001"},{"key":"2025071508501003000_2025.07.10.663928v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.05.015"},{"key":"2025071508501003000_2025.07.10.663928v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/S1535-6108(03)00110-7"},{"key":"2025071508501003000_2025.07.10.663928v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2004.03.027"},{"key":"2025071508501003000_2025.07.10.663928v1.40","doi-asserted-by":"publisher","DOI":"10.1126\/science.1140321"},{"key":"2025071508501003000_2025.07.10.663928v1.41","doi-asserted-by":"publisher","DOI":"10.1126\/science.1083430"},{"key":"2025071508501003000_2025.07.10.663928v1.42","doi-asserted-by":"publisher","DOI":"10.1038\/nrm.2017.67"},{"key":"2025071508501003000_2025.07.10.663928v1.43","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky020"},{"key":"2025071508501003000_2025.07.10.663928v1.44","doi-asserted-by":"publisher","DOI":"10.1093\/narcan\/zcad006"},{"key":"2025071508501003000_2025.07.10.663928v1.45","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1610011114"},{"key":"2025071508501003000_2025.07.10.663928v1.46","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1301445110"},{"key":"2025071508501003000_2025.07.10.663928v1.47","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2013.5668"},{"key":"2025071508501003000_2025.07.10.663928v1.48","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M212360200"},{"key":"2025071508501003000_2025.07.10.663928v1.49","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-06-4328"},{"key":"2025071508501003000_2025.07.10.663928v1.50","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2007.01.018"},{"key":"2025071508501003000_2025.07.10.663928v1.51","doi-asserted-by":"publisher","DOI":"10.1080\/02656730802631775"},{"key":"2025071508501003000_2025.07.10.663928v1.52","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-08-1639"},{"key":"2025071508501003000_2025.07.10.663928v1.53","doi-asserted-by":"publisher","DOI":"10.3791\/56617"},{"key":"2025071508501003000_2025.07.10.663928v1.54","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1416031112"},{"key":"2025071508501003000_2025.07.10.663928v1.55","doi-asserted-by":"publisher","DOI":"10.2174\/1871520619666190305141458"},{"key":"2025071508501003000_2025.07.10.663928v1.56","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a012583"},{"key":"2025071508501003000_2025.07.10.663928v1.57","doi-asserted-by":"publisher","DOI":"10.1016\/j.dnarep.2017.02.010"},{"key":"2025071508501003000_2025.07.10.663928v1.58","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-49519-z"},{"key":"2025071508501003000_2025.07.10.663928v1.59","doi-asserted-by":"publisher","DOI":"10.1002\/em.20430"},{"key":"2025071508501003000_2025.07.10.663928v1.60","doi-asserted-by":"publisher","DOI":"10.1016\/j.mrgentox.2009.07.007"},{"key":"2025071508501003000_2025.07.10.663928v1.61","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.200612031"},{"key":"2025071508501003000_2025.07.10.663928v1.62","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.200608077"},{"key":"2025071508501003000_2025.07.10.663928v1.63","doi-asserted-by":"publisher","DOI":"10.1016\/S0065-2776(08)00602-0"},{"key":"2025071508501003000_2025.07.10.663928v1.64","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0406466102"},{"key":"2025071508501003000_2025.07.10.663928v1.65","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2015.02.024"},{"key":"2025071508501003000_2025.07.10.663928v1.66","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20020973"},{"key":"2025071508501003000_2025.07.10.663928v1.67","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2022.167858"},{"key":"2025071508501003000_2025.07.10.663928v1.68","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2021.738502"},{"key":"2025071508501003000_2025.07.10.663928v1.69","doi-asserted-by":"publisher","DOI":"10.1186\/1478-811X-8-31"},{"key":"2025071508501003000_2025.07.10.663928v1.70","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac853"},{"key":"2025071508501003000_2025.07.10.663928v1.71","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2023.03.017"},{"key":"2025071508501003000_2025.07.10.663928v1.72","doi-asserted-by":"publisher","DOI":"10.1038\/s41408-018-0129-9"},{"key":"2025071508501003000_2025.07.10.663928v1.73","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-07-0484"},{"key":"2025071508501003000_2025.07.10.663928v1.74","doi-asserted-by":"publisher","DOI":"10.1016\/j.canlet.2021.08.030"},{"key":"2025071508501003000_2025.07.10.663928v1.75","doi-asserted-by":"publisher","DOI":"10.1093\/carcin\/12.10.1857"},{"key":"2025071508501003000_2025.07.10.663928v1.76","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2011.08.039"},{"key":"2025071508501003000_2025.07.10.663928v1.77","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2016.10.001"},{"key":"2025071508501003000_2025.07.10.663928v1.78","doi-asserted-by":"publisher","DOI":"10.1038\/nature24484"},{"key":"2025071508501003000_2025.07.10.663928v1.79","doi-asserted-by":"publisher","DOI":"10.1002\/jcp.25048"},{"key":"2025071508501003000_2025.07.10.663928v1.80","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.131009198"},{"key":"2025071508501003000_2025.07.10.663928v1.81","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-03159-2"},{"key":"2025071508501003000_2025.07.10.663928v1.82","doi-asserted-by":"publisher","DOI":"10.3791\/55745"},{"key":"2025071508501003000_2025.07.10.663928v1.83","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.105032"},{"key":"2025071508501003000_2025.07.10.663928v1.84","doi-asserted-by":"publisher","DOI":"10.3892\/mmr.2019.10640"},{"key":"2025071508501003000_2025.07.10.663928v1.85","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2011.02.013"},{"key":"2025071508501003000_2025.07.10.663928v1.86","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA119.010497"},{"key":"2025071508501003000_2025.07.10.663928v1.87","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2380"},{"key":"2025071508501003000_2025.07.10.663928v1.88","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki749"},{"key":"2025071508501003000_2025.07.10.663928v1.89","doi-asserted-by":"publisher","DOI":"10.1093\/carcin\/bgm157"},{"key":"2025071508501003000_2025.07.10.663928v1.90","doi-asserted-by":"publisher","DOI":"10.1126\/science.1237150"},{"key":"2025071508501003000_2025.07.10.663928v1.91","doi-asserted-by":"publisher","DOI":"10.1038\/nature02118"},{"key":"2025071508501003000_2025.07.10.663928v1.92","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04767-1"},{"key":"2025071508501003000_2025.07.10.663928v1.93","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa718"},{"key":"2025071508501003000_2025.07.10.663928v1.94","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2024.107337"},{"key":"2025071508501003000_2025.07.10.663928v1.95","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkz1175"},{"key":"2025071508501003000_2025.07.10.663928v1.96","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv1520"},{"key":"2025071508501003000_2025.07.10.663928v1.97","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2089"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.07.10.663928","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T09:06:34Z","timestamp":1768467994000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.07.10.663928"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,12]]},"references-count":97,"URL":"https:\/\/doi.org\/10.1101\/2025.07.10.663928","relation":{},"subject":[],"published":{"date-parts":[[2025,7,12]]},"subtype":"preprint"}}