title: Telomere Chromatin Condensation Assay (TCCA): a novel approach to study structural telomere integrity.
abstract: Telomeres, the DNA-protein complexes located at the end of linear eukaryotic chromosomes are essential for genome stability. Improper higher-order chromatin organization at the chromosome ends can give rise to telomeric recombination and genomic instability. We report the development of an assay to quantify differences in the condensation of telomeric chromatin, thereby offering new opportunities to study telomere biology and stability. We have combined a DNA nuclease digestion with a quantitative PCR (qPCR) assay of telomeric DNA, which we term the Telomere Chromatin Condensation Assay (TCCA). By quantifying the relative quantities of telomeric DNA that are progressively digested with the exonuclease Bal 31 the method can discriminate between different levels of telomeric chromatin condensation. The structural chromatin packaging at telomeres shielded against exonuclease digestion delivered an estimate, which we term Chromatin Protection Factor (CPF) that ranged from 1.7 to 2.3 fold greater than that present in unpacked DNA. The CPF was significantly decreased when cell cultures were incubated with the DNA hypomethylating agent 5-azacytidine, demonstrating the ability of the TCCA assay to discriminate between packaging levels of telomeric DNA.
authors.affiliation: INIBIC-Complejo Hospitalario Universitario A Coruña, Unidad de Genética, As Xubias, 84, 15006 A Coruña, Spain.
authors.forename: Iria
authors.initials: I
authors.lastname: Gonzalez-Vasconcellos
authors.affiliation: INIBIC-Complejo Hospitalario Universitario A Coruña, Unidad de Genética, As Xubias, 84, 15006 A Coruña, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Alonso-Rodríguez
authors.affiliation: Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, 15009 A Coruña, Spain.
authors.forename: Isidoro
authors.initials: I
authors.lastname: López-Baltar
authors.affiliation: INIBIC-Complejo Hospitalario Universitario A Coruña, Unidad de Genética, As Xubias, 84, 15006 A Coruña, Spain; Laboratorio de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, 15009 A Coruña, Spain. Electronic address: jose.luis.fernandez.garcia@sergas.es.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Fernández
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000473:pathology; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods*; D016615:Telomere / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D002843:Chromatin; D004247:DNA; D003851:Deoxyribonucleases; D001374:Azacitidine
keywords: Bal 31 nuclease; CPF (Chromatin protection factor); Chromatin condensation; Enzymatic digestion; TCCA (telomere chromatin condensation assay); Telomere integrity