title: Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body.
abstract: Telomere maintenance is essential for continued cell proliferation. Although most cells accomplish this by activating telomerase, a subset of immortalized tumors and cell lines do so in a telomerase-independent manner, a process called alternative lengthening of telomeres (ALT). DNA recombination has been shown to be involved in ALT, but the precise mechanisms remain unknown. A fraction of cells in a given ALT population contain a unique nuclear structure called APB (ALT-associated promyelocytic leukemia (PML) body), which is characterized by the presence of telomeric DNA in the PML body. Here we describe that hRad9, hHus1, and hRad1, which form a DNA clamp complex that is associated with DNA damage, as well as its clamp loader, hRad17, are constitutive components of APB. Phosphorylated histone H2AX (gamma-H2AX), a molecular marker of double-strand breaks (DSBs), also colocalizes with some APBs. The results suggest that telomeric DNAs at APBs are recognized as DSBs. PML staining and fluorescence in situ hybridization analyses of mitotic ALT cells revealed that telomeric DNAs present at APBs are of both extrachromosomal and native telomere origins. Furthermore, we demonstrated that DNA synthesis occurs at APBs and is significantly inhibited by caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases. Taken together, we suggest that telomeric DNAs at APBs are recognized and processed as DSBs, leading to telomeric DNA synthesis and thereby contributing to telomere maintenance in ALT cells.
authors.affiliation: Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Kyoto 606-8502.
authors.forename: Akira
authors.initials: A
authors.lastname: Nabetani
authors.forename: Osamu
authors.initials: O
authors.lastname: Yokoyama
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Ishikawa
mesh_terms: D001973:Bromodeoxyuridine / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000502:physiology; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins; D005092:Exonucleases / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis*; D009687:Nuclear Proteins / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D016615:Telomere; D014157:Transcription Factors / Q000032:analysis*; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C462949:HUS1B protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C113575:Rad17 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C069930:rad9 protein; D002110:Caffeine; D017346:Protein Serine-Threonine Kinases; D005092:Exonucleases; C113574:Rad1 protein, human; D001973:Bromodeoxyuridine