title: Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells.
abstract: In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.
authors.affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-Cho, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Yugo
authors.initials: Y
authors.lastname: Hirai
authors.forename: Kenkichi
authors.initials: K
authors.lastname: Masutomi
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Ishikawa
mesh_terms: D001973:Bromodeoxyuridine / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016196:S Phase / Q000235:genetics; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*; D016615:Telomere / Q000235:genetics*
chemical_list: D002843:Chromatin; D004277:DNA, Single-Stranded; C509186:TERT protein, human; D019098:Telomerase; D001973:Bromodeoxyuridine