biostudies-literatureGerodimos CANCI NIH HHSNational Institutes of HealthNIGMS NIH HHS103193https://www.ebi.ac.uk/biostudies/studies/S-EPMC8435023biostudies-literatureUnknown106The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligasDNA repairNonhomologous DNA end joining of nucleosomal substrates in a purified system.PMC8435023R01 CA100504R35 GM118009CA100504GM118009Watanabe GGerodimos CALieber MRfalseNonhomologous DNA end joining of nucleosomal substrates in a purified system.The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligas2021-01-01T00:00:00Z2021 Oct2025-04-04T12:04:27.004Z2025-04-04T12:04:27.004ZS-EPMC84350233433994810.1016/j.dnarep.2021.103193