{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T05:26:40Z","timestamp":1786685200839,"version":"build-2736575974"},"reference-count":48,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100001340","name":"W. W. Smith Charitable Trust","doi-asserted-by":"publisher","award":["#C2007"],"award-info":[{"award-number":["#C2007"]}],"id":[{"id":"10.13039\/100001340","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001340","name":"W. W. Smith Charitable Trust","doi-asserted-by":"publisher","award":["#C2106"],"award-info":[{"award-number":["#C2106"]}],"id":[{"id":"10.13039\/100001340","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100001340","name":"W. W. Smith Charitable Trust","doi-asserted-by":"publisher","award":["NIH\/NIA AG071815"],"award-info":[{"award-number":["NIH\/NIA AG071815"]}],"id":[{"id":"10.13039\/100001340","id-type":"DOI","asserted-by":"publisher"}]},{"award":["#C2007"],"award-info":[{"award-number":["#C2007"]}],"id":[{"id":"https:\/\/ror.org\/04nv2ya05","id-type":"ROR","asserted-by":"publisher"}]},{"award":["#C2106"],"award-info":[{"award-number":["#C2106"]}],"id":[{"id":"https:\/\/ror.org\/04nv2ya05","id-type":"ROR","asserted-by":"publisher"}]},{"award":["NIH\/NIA AG071815"],"award-info":[{"award-number":["NIH\/NIA AG071815"]}],"id":[{"id":"https:\/\/ror.org\/04nv2ya05","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Pennsylvania Department of Health Formula"},{"id":[{"id":"https:\/\/ror.org\/00ra1fg11","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008212","name":"Drexel University College of Medicine","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100008212","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/04bdffz58","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Timeless and its fission yeast ortholog Swi1 are evolutionarily conserved components of the replication fork protection complex that ensures faithful DNA replication and genome stability. While their nuclear roles are well-characterized, their roles in mitochondrial genome maintenance remain unknown. Here, we demonstrate a previously unrecognized connection between Timeless\/Swi1 and mitochondrial homeostasis. In fission yeast, swi1 deletion increased association of the DNA repair protein Rad52 with mitochondrial DNA sequences across the mitochondrial genome, suggesting altered mitochondrial genome maintenance. Unexpectedly, swi1\u2206 cells showed an increased mtDNA copy number and improved growth under respiratory conditions, suggesting activation of compensatory mechanisms that promote mitochondrial genome maintenance. The loss of Swi1 also partially rescued the growth defect under respiratory conditions and mtDNA loss associated with depletion of mitochondrial DNA polymerase \u03b3, linking Swi1 to pathways regulating mitochondrial replication under stress. Consistent with these phenotypes, transcriptomic and pathway enrichment analyses revealed transcriptional changes indicative of reduced glycolysis and enhanced oxidative phosphorylation, suggesting a shift toward respiratory metabolism. In human cells, Timeless depletion elicited distinct mitochondrial responses depending on the cell type. While Timeless-depleted TE-11 and Saos-2 cells elicited mitochondrial phenotypes comparable to those observed in fission yeast, Timeless depletion in U-2 OS cells led to reduced mtDNA copy number, elevated mitochondrial reactive oxygen species, and decreased mitochondrial membrane potential and mass, consistent with mitochondrial dysfunction. Despite these phenotypic differences, both fission yeast and human cells exhibited elevated levels of orthologs of the mitochondrial transcription factor A (TFAM) and the oxidative stress regulator NRF2, suggesting the conserved activation of compensatory mitochondrial and antioxidant pathways. Together, these findings identify an evolutionarily conserved connection between Timeless\/Swi1 and mitochondrial homeostasis and reveal distinct adaptive responses to mitochondrial stress.<\/jats:p>","DOI":"10.3390\/biom16081177","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T11:02:11Z","timestamp":1786532531000},"page":"1177","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evolutionarily Conserved but Mechanistically Distinct Mitochondrial Responses to Loss of Timeless\/Swi1"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6524-4223","authenticated-orcid":false,"given":"Kalisse I.","family":"Horne","sequence":"first","affiliation":[{"name":"Program in Molecular and Cell Biology and Genetics, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, PA 19102, USA"},{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua Chang","family":"Mell","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6924-7415","authenticated-orcid":false,"given":"Chiaki","family":"Noguchi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0121-6301","authenticated-orcid":false,"given":"Sri Havya","family":"Jana","sequence":"additional","affiliation":[{"name":"Program in Molecular and Cell Biology and Genetics, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, PA 19102, USA"},{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shriya","family":"Pinisetty","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rhea","family":"Masand","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Sell","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5470-5127","authenticated-orcid":false,"given":"Eishi","family":"Noguchi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3945","DOI":"10.4161\/cc.21989","article-title":"Local and global functions of Timeless and Tipin in replication fork protection","volume":"11","author":"Leman","year":"2012","journal-title":"Cell Cycle"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1007\/s00018-023-04738-3","article-title":"The TIMELESS effort for timely DNA replication and protection","volume":"80","author":"Patel","year":"2023","journal-title":"Cell Mol. Life Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"168206","DOI":"10.1016\/j.jmb.2023.168206","article-title":"The TIMELESS Roles in Genome Stability and Beyond","volume":"436","author":"Vipat","year":"2024","journal-title":"J. Mol. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Gadaleta, M.C., Das, M.M., Tanizawa, H., Chang, Y.T., Noma, K., Nakamura, T.M., and Noguchi, E. (2016). Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres. PLoS Genet., 12.","DOI":"10.1371\/journal.pgen.1005943"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Gadaleta, M.C., and Noguchi, E. (2017). Regulation of DNA Replication through Natural Impediments in the Eukaryotic Genome. Genes, 8.","DOI":"10.3390\/genes8030098"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1038\/s41392-023-01547-9","article-title":"Mitochondrial dynamics in health and disease: Mechanisms and potential targets","volume":"8","author":"Chen","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e36470","DOI":"10.1016\/j.heliyon.2024.e36470","article-title":"Molecular mechanisms of mitochondrial homeostasis regulation in neurons and possible therapeutic approaches for Alzheimer\u2019s disease","volume":"10","author":"Ren","year":"2024","journal-title":"Heliyon"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2601","DOI":"10.1016\/j.cell.2024.04.037","article-title":"Mitochondria at the crossroads of health and disease","volume":"187","author":"Suomalainen","year":"2024","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Rong, Z., Tu, P., Xu, P., Sun, Y., Yu, F., Tu, N., Guo, L., and Yang, Y. (2021). The Mitochondrial Response to DNA Damage. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.669379"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1128\/MMBR.00007-13","article-title":"Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA","volume":"77","author":"Chen","year":"2013","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1038\/s42003-021-01647-8","article-title":"FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability","volume":"4","author":"Fernandes","year":"2021","journal-title":"Commun. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Mochizuki-Kashio, M., Otsuki, N., Fujiki, K., Abdelhamd, S., Kurre, P., Grompe, M., Iwama, A., Saito, K., and Nakamura-Ishizu, A. (2023). Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells. Front. Oncol., 13.","DOI":"10.3389\/fonc.2023.1108430"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"18984","DOI":"10.1074\/jbc.M109.099846","article-title":"Discovery of a novel function for human Rad51: Maintenance of the mitochondrial genome","volume":"285","author":"Sage","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/j.mito.2013.04.004","article-title":"Human Rad51 promotes mitochondrial DNA synthesis under conditions of increased replication stress","volume":"13","author":"Sage","year":"2013","journal-title":"Mitochondrion"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4266","DOI":"10.1105\/tpc.111.091744","article-title":"Identification of plant RAD52 homologs and characterization of the Arabidopsis thaliana RAD52-like genes","volume":"23","author":"Samach","year":"2011","journal-title":"Plant Cell"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Stein, A., Kalifa, L., and Sia, E.A. (2015). Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae. PLoS Genet., 11.","DOI":"10.1371\/journal.pgen.1005664"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"976","DOI":"10.1002\/1873-3468.14021","article-title":"Mitochondrial DNA copy number in human disease: The more the better?","volume":"595","author":"Filograna","year":"2021","journal-title":"FEBS Lett."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Hagan, I.M., Carr, A.M., Grallert, A., and Nurse, P. (2016). Fission Yeast: A Laboratory Manual, Cold Spring Harbor Laboratory Press.","DOI":"10.1101\/pdb.prot091033"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1280","DOI":"10.1002\/biot.200800140","article-title":"A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe","volume":"3","author":"Noguchi","year":"2008","journal-title":"Biotechnol. J."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Roseaulin, L.C., Noguchi, C., Martinez, E., Ziegler, M.A., Toda, T., and Noguchi, E. (2013). Coordinated degradation of replisome components ensures genome stability upon replication stress in the absence of the replication fork protection complex. PLoS Genet., 9.","DOI":"10.1371\/journal.pgen.1003213"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"257","DOI":"10.2144\/000114100","article-title":"New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe","volume":"55","author":"Gadaleta","year":"2013","journal-title":"Biotechniques"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1080\/15384101.2016.1237326","article-title":"Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast","volume":"16","author":"Noguchi","year":"2017","journal-title":"Cell Cycle"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e13068","DOI":"10.1111\/acel.13068","article-title":"Maf1-dependent transcriptional regulation of tRNAs prevents genomic instability and is associated with extended lifespan","volume":"19","author":"Shetty","year":"2020","journal-title":"Aging Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"114828","DOI":"10.1016\/j.yexcr.2025.114828","article-title":"Timeless prevents senescence-associated phenotypes and enhances DNA repair to promote esophageal cancer cell growth","volume":"455","author":"Noguchi","year":"2026","journal-title":"Exp. Cell Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3109","DOI":"10.1002\/1878-0261.13072","article-title":"FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes","volume":"15","author":"Peake","year":"2021","journal-title":"Mol. Oncol."},{"key":"ref_26","unstructured":"Burke, D., Dawson, D., and Stearns, T. (2000). Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1007\/978-1-4939-2596-4_12","article-title":"Chromatin immunoprecipitation to detect DNA replication and repair factors","volume":"1300","author":"Gadaleta","year":"2015","journal-title":"Methods Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1038\/nature724","article-title":"The genome sequence of Schizosaccharomyces pombe","volume":"415","author":"Wood","year":"2002","journal-title":"Nature"},{"key":"ref_29","unstructured":"Li, H. (2026, July 26). Aligning Sequence Reads, Clone Sequences and Assembly Contigs with BWA-MEM. Available online: https:\/\/arxiv.org\/abs\/1303.3997."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2032","DOI":"10.1093\/bioinformatics\/btv098","article-title":"Sambamba: Fast processing of NGS alignment formats","volume":"31","author":"Tarasov","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"R137","DOI":"10.1186\/gb-2008-9-9-r137","article-title":"Model-based analysis of ChIP-Seq (MACS)","volume":"9","author":"Zhang","year":"2008","journal-title":"Genome Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1038\/nbt.1754","article-title":"Integrative genomics viewer","volume":"29","author":"Robinson","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1093\/bioinformatics\/btq033","article-title":"BEDTools: A flexible suite of utilities for comparing genomic features","volume":"26","author":"Quinlan","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Lawrence, M., Huber, W., Pag\u00e8s, H., Aboyoun, P., Carlson, M., Gentleman, R., Morgan, M.T., and Carey, V.J. (2013). Software for computing and annotating genomic ranges. PLoS Comput. Biol., 9.","DOI":"10.1371\/journal.pcbi.1003118"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1242\/jcs.057984","article-title":"Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion","volume":"123","author":"Leman","year":"2010","journal-title":"J. Cell Sci."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Nacarelli, T., Azar, A., and Sell, C. (2014). Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion. Front. Endocrinol., 5.","DOI":"10.3389\/fendo.2014.00122"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1534\/genetics.105.046144","article-title":"Capture of extranuclear DNA at fission yeast double-strand breaks","volume":"171","author":"Decottignies","year":"2005","journal-title":"Genetics"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1250","DOI":"10.1101\/gr.104513.109","article-title":"Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe","volume":"20","author":"Lenglez","year":"2010","journal-title":"Genome Res."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Chu, Z., Li, J., Eshaghi, M., Karuturi, R.K., Lin, K., and Liu, J. (2007). Adaptive expression responses in the Pol-gamma null strain of S. pombe depleted of mitochondrial genome. BMC Genom., 8.","DOI":"10.1186\/1471-2164-8-323"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/j.bbadis.2010.11.012","article-title":"POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts","volume":"1812","author":"Stewart","year":"2011","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1038\/s41586-025-08856-9","article-title":"Small molecules restore mutant mitochondrial DNA polymerase activity","volume":"642","author":"Valenzuela","year":"2025","journal-title":"Nature"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Lin, L., Wu, Q., Lu, F., Lei, J., Zhou, Y., Liu, Y., Zhu, N., Yu, Y., Ning, Z., and She, T. (2023). Nrf2 signaling pathway: Current status and potential therapeutic targetable role in human cancers. Front. Oncol., 13.","DOI":"10.3389\/fonc.2023.1184079"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1673","DOI":"10.1016\/j.biochi.2006.03.001","article-title":"Regulation of the cadmium stress response through SCF-like ubiquitin ligases: Comparison between Saccharomyces cerevisiae, Schizosaccharomyces pombe and mammalian cells","volume":"88","author":"Labarre","year":"2006","journal-title":"Biochimie"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1093\/toxsci\/kfs179","article-title":"Cadmium-induced proteome remodeling regulated by Spc1\/Sty1 and Zip1 in fission yeast","volume":"129","author":"Guo","year":"2012","journal-title":"Toxicol. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"7861","DOI":"10.1128\/MCB.23.21.7861-7874.2003","article-title":"Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1","volume":"23","author":"Noguchi","year":"2003","journal-title":"Mol. Cell Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"8342","DOI":"10.1128\/MCB.24.19.8342-8355.2004","article-title":"Swi1 and Swi3 are components of a replication fork protection complex in fission yeast","volume":"24","author":"Noguchi","year":"2004","journal-title":"Mol. Cell Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1549","DOI":"10.1038\/s41594-023-01091-8","article-title":"Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth","volume":"30","author":"Seel","year":"2023","journal-title":"Nat. Struct. Mol. Biol."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/8\/1177\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T04:26:59Z","timestamp":1786681619000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/8\/1177"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,12]]},"references-count":48,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biom16081177"],"URL":"https:\/\/doi.org\/10.3390\/biom16081177","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,12]]}}}