{"doi":"10.1093/nar/gkaf711","title":"ecDNA replication is disorganized and vulnerable to replication stress","abstract":"<jats:title>Abstract</jats:title>\n               <jats:p>Extrachromosomal DNA (ecDNA) is a critical driver of cancer progression, contributing to tumour growth, evolution, and therapeutic resistance through oncogene amplification. Despite its significance, the replication of ecDNA remains poorly understood. In this study, we investigated the replication dynamics of ecDNA using high-resolution replication timing analysis (Repli-seq) and DNAscent, a method for measuring origin firing and replication fork movement, that we applied to both bulk DNA and to ecDNA isolated with FINE (Fluorescence-activated cell sorting-based Isolation of Native ecDNA), a new method for isolating, chromatinized ecDNA without DNA or protein digestion. We demonstrate that ecDNA in the COLO 320DM colorectal cancer cell line exhibits largely asynchronous replication throughout the S phase, contrasting with the conserved replication timing of the corresponding chromosomal DNA in RPE-1 cells and the chromosomally reintegrated ecDNA in COLO 320HSR. Replication origins on ecDNA are redistributed, and replication forks exhibit reduced velocity and increased stalling. Under replication stress induced by hydroxyurea treatment, ecDNA replication is further compromised, leading to altered origin activation, reduced fork velocity and eventual ecDNA depletion from cells. Our findings reveal fundamental differences in the replication dynamics of ecDNA, providing insights that could inform the development of therapies targeting ecDNA-associated oncogene amplification in cancer.</jats:p>","journal":"Nucleic Acids Research","year":2025,"id":598255,"datarank":0.40620753016533157,"base_score":2.70805020110221,"endowment":2.70805020110221,"self_citation_contribution":0.40620753016533157,"citation_network_contribution":0.0,"self_endowment_contribution":0.40620753016533157,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":14,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1532889,"name":"Pauline L Pfuderer","orcid":null,"position":1,"is_corresponding":false},{"id":1532890,"name":"Pawel Czyz","orcid":null,"position":2,"is_corresponding":false},{"id":1057373,"name":"Gianluca Petris","orcid":"0000-0002-2420-6359","position":3,"is_corresponding":false},{"id":1532891,"name":"Michael A Boemo","orcid":null,"position":4,"is_corresponding":false},{"id":600824,"name":"Julian E. Sale","orcid":"0000-0002-5031-3780","position":5,"is_corresponding":false},{"id":1532888,"name":"Jedrzej J Jaworski","orcid":"0000-0002-7680-4801","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"ecDNA replication is disorganized and vulnerable to replication stress","abstract":"<jats:title>Abstract</jats:title>\n               <jats:p>Extrachromosomal DNA (ecDNA) is a critical driver of cancer progression, contributing to tumour growth, evolution, and therapeutic resistance through oncogene amplification. Despite its significance, the replication of ecDNA remains poorly understood. In this study, we investigated the replication dynamics of ecDNA using high-resolution replication timing analysis (Repli-seq) and DNAscent, a method for measuring origin firing and replication fork movement, that we applied to both bulk DNA and to ecDNA isolated with FINE (Fluorescence-activated cell sorting-based Isolation of Native ecDNA), a new method for isolating, chromatinized ecDNA without DNA or protein digestion. We demonstrate that ecDNA in the COLO 320DM colorectal cancer cell line exhibits largely asynchronous replication throughout the S phase, contrasting with the conserved replication timing of the corresponding chromosomal DNA in RPE-1 cells and the chromosomally reintegrated ecDNA in COLO 320HSR. Replication origins on ecDNA are redistributed, and replication forks exhibit reduced velocity and increased stalling. Under replication stress induced by hydroxyurea treatment, ecDNA replication is further compromised, leading to altered origin activation, reduced fork velocity and eventual ecDNA depletion from cells. Our findings reveal fundamental differences in the replication dynamics of ecDNA, providing insights that could inform the development of therapies targeting ecDNA-associated oncogene amplification in cancer.</jats:p>","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"40744497","pmcid":"PMC12311790","openalex_id":null,"authors":[],"funders":[{"funder_name":"MRC","grant_id":"MC_U105178808","title":"Vertebrate mutagenesis and DNA damage tolerance"},{"funder_name":"Cancer Research UK Cambridge Centre","grant_id":"C9685/A25117","title":null},{"funder_name":"Engineering and Physical Sciences Research Council","grant_id":"EP/T022159/1","title":"Cambridge Service for Data Driven Discovery (CSD3) - A National Data Intensive Science Cloud for Converged Simulation, AI &amp; Analytics"},{"funder_name":"Marie Skłodowska-Curie European Postdoctoral Fellowship","grant_id":"897663","title":"GENome Editing and delivery Strategies for REcoding the mammalian genome"},{"funder_name":"Dell EMC","grant_id":"","title":null},{"funder_name":"Science and Technology Facilities Council","grant_id":"","title":null},{"funder_name":"Boehringer Ingelheim Fonds","grant_id":"","title":null},{"funder_name":"Cambridge Service for Data Driven Discovery","grant_id":"","title":null},{"funder_name":"University of Cambridge Research Computing Service","grant_id":"","title":null},{"funder_name":"Associate Faculty Member of the Wellcome Sanger Institute","grant_id":"","title":null},{"funder_name":"ETH AI Centre","grant_id":"","title":null},{"funder_name":"Associate Faculty Member of the Wellcome Sanger Institute","grant_id":"","title":null},{"funder_name":"Boehringer Ingelheim Fonds","grant_id":"","title":null},{"funder_name":"ETH AI Centre","grant_id":"","title":null},{"funder_name":"Cambridge Service for Data Driven Discovery","grant_id":"","title":null},{"funder_name":"University of Cambridge Research Computing Service","grant_id":"","title":null},{"funder_name":"Dell EMC","grant_id":"","title":null},{"funder_name":"Science and Technology Facilities Council","grant_id":"","title":null}],"total_grants":18,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://academic.oup.com/nar/article-pdf/53/14/gkaf711/63899917/gkaf711.pdf","host_type":"publisher"},{"url":"https://hdl.handle.net/11390/1312540","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/12311790","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC12311790","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC12311790?pdf=render","host_type":"Europe_PMC"},{"url":"https://doi.org/10.1101/2025.03.22.644567","host_type":""},{"url":"https://doi.org/10.1093/nar/gkaf711","host_type":""},{"url":"https://dx.doi.org/10.3929/ethz-c-000782763","host_type":""},{"url":"https://pubmed.ncbi.nlm.nih.gov/40744497","host_type":""},{"url":"http://dx.doi.org/10.1093/nar/gkaf711","host_type":""}],"fields_of_study":["0301 basic medicine","03 medical and health sciences","Humans","DNA Replication","Cell Line, Tumor","Hydroxyurea","Replication Origin","DNA","Colorectal Neoplasms","DNA Replication Timing","S Phase","Stress, Physiological"],"mesh_terms":["Cell Line, Tumor","Humans","Colorectal Neoplasms","Hydroxyurea","DNA","S Phase","DNA Replication","DNA Replication Timing","Replication Origin","Stress, Physiological"],"keywords":["DNA Replication","DNA Replication Timing","Stress, Physiological","Cell Line, Tumor","Humans","Hydroxyurea","Replication Origin","DNA","Genome Integrity, Repair and Replication","Colorectal Neoplasms","S Phase"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[{"name":"bioproject"}],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-28T15:22:08.233063Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}