Supplementary Table 2 from Telomere Dysfunction and DNA-PKcs Deficiency: Characterization and Consequence

Supplementary Table 1 from Telomere Dysfunction and DNA-PKcs Deficiency: Characterization and Consequence

Abstract The mechanisms by which cells accurately distinguish between DNA double-strand break (DSB) ends and telomeric DNA ends remain poorly defined. Recent investigations have revealed intriguing interactions between DNA repair and telomeres. We were the first to report a requirement for the nonhomologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PK) in the effective end-capping of mammalian telomeres. Here, we report our continued characterization of uncapped (as opposed to shortened) dysfunctional telomeres in cells deficient for the catalytic subunit of DNA-PK (DNA-PKcs) and shed light on their consequence. We present evidence in support of our model that uncapped telomeres in this repair-deficient background are inappropriately detected and processed as DSBs and thus participate not only in spontaneous telomere-telomere fusion but, importantly, also in ionizing radiation–induced telomere-DSB fusion events. We show that phosphorylation of DNA-PKcs itself (Thr-2609 cluster) is a critical event for proper telomere end-processing and that ligase IV (NHEJ) is required for uncapped telomere fusion. We also find uncapped telomeres in cells from the BALB/c mouse, which harbors two single-nucleotide polymorphisms that result in reduced DNA-PKcs abundance and activity, most markedly in mammary tissue, and are both radiosensitive and susceptible to radiogenic mammary cancer. Our results suggest mechanistic links between uncapped/dysfunctional telomeres in DNA-PKcs–deficient backgrounds, radiation-induced instability, and breast cancer. These studies provide the first direct evidence of genetic susceptibility and environmental insult interactions leading to a unique and ongoing form of genomic instability capable of driving carcinogenesis. [Cancer Res 2009;69(5):2100–7]

Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies.

ABC Fax + 41 61 306 12 34 E-mail karger@karger.ch www.karger.com © 2003 S. Karger AG, Basel 0301–0171/02/0984–0265$19.50/0 Accessible online at: www.karger.com/cgr Abstract. The microchromosomes (MICs) in chicken DT40 lymphocytes are usually clustered in the center of the nucleus, whereas the macrochromosomes (MACs) are preferentially located toward the nuclear periphery. This compartmentalized architecture of the nucleus is associated with a low frequency of translocations between MICs and MACs after induction of DNA breaks by a radiation track(s). In contrast, the MICs in chick embryo fibroblasts (CEFs) tend to be located throughout the entire nuclear volume. The resulting side-to-side arrangement of MIC and MAC territories favors radiation-induced MIC/MAC translocations, which occur more frequently in CEF cells than MIC/MIC or MAC/MAC rearrangements. Collectively, our results suggest that preformed physical contacts are a prerequisite for the generation of chromosome rearrangements through recombinational repair of DNA damage. Cell type-specific higher-order nuclear organization may prevent or stimulate the formation of particular chromosome aberrations in pathology and evolution. Ectopic expression of the recombination protein Rad51 can protect cells from radiation-induced translocations. The repair activity of overexpressed Rad51 is more important for cells that are irradiated in S/G2 phase than for cells in G1 phase. Evidently, homologous recombination between sister chromatids of a replicated chromosome is more frequent than that between homologous or heterologous chromosomes during G1 phase.

The microchromosomes (MICs) in chicken DT40 lymphocytes are usually clustered in the center of the nucleus, whereas the macrochromosomes (MACs) are preferentially located toward the nuclear periphery. This compartmentalized architecture of the nucleus is associated with a low frequency of translocations between MICs and MACs after induction of DNA breaks by a radiation track(s). In contrast, the MICs in chick embryo fibroblasts (CEFs) tend to be located throughout the entire nuclear volume. The resulting side-to-side arrangement of MIC and MAC territories favors radiation-induced MIC/MAC translocations, which occur more frequently in CEF cells than MIC/MIC or MAC/MAC rearrangements. Collectively, our results suggest that preformed physical contacts are a prerequisite for the generation of chromosome rearrangements through recombinational repair of DNA damage. Cell type-specific higher-order nuclear organization may prevent or stimulate the formation of particular chromosome aberrations in pathology and evolution. Ectopic expression of the recombination protein Rad51 can protect cells from radiation-induced translocations. The repair activity of overexpressed Rad51 is more important for cells that are irradiated in S/G2 phase than for cells in G1 phase. Evidently, homologous recombination between sister chromatids of a replicated chromosome is more frequent than that between homologous or heterologous chromosomes during G1 phase.

Editor—Carriers of de novo balanced reciprocal translocations and inversions have an increased risk of approximately 6% for developing multiple congenital abnormalities (MCA) and/or mental retardation (MR), compared to a 2-3% risk overall in newborn populations.1 2 Cytogenetically cryptic deletions or physical disruption or inactivation of a gene(s) in one or both breakpoint regions may account for the observed phenotypes.3 4 It seems plausible to assume that the risk for MCA/MR may be even higher in carriers of de novo complex chromosome rearrangements (CCRs), which involve at least three different chromosomes and breakpoint regions. Extreme cases involving up to seven chromosomes and 10 breakpoints have been described.5-7Indeed, most reported CCRs are associated with MCA/MR.8 9In addition, they have been found in infertile men10 and in women suffering from multiple miscarriages.11 12 The complex nature of CCRs renders karyotype interpretation by classical chromosome banding alone difficult. In many cases fluorescence in situ hybridisation (FISH) will be the best method to delineate the underlying chromosome rearrangements.7 13Here we have applied conventional FISH with chromosome painting probes and region specific large insert clones, comparative genomic hybridisation (CGH),14 15 and spectral karyotyping (SKY)16 17 to an apparently balanced and very complex rearrangement in a profoundly retarded patient with Moebius syndrome (MBS, MIM 157900).18 MBS is characterised by congenital paralysis of the seventh cranial nerve leading to facial diplegia.19 Other cranial nerves may also be affected. In addition, orofacial and limb malformations, defects of the musculoskeletal system, and MR may occur. This patient, who has classical Moebius syndrome, has been reported previously.4 He was the third child born to a 35 year old father and a 32 year old mother. Because of paresis of the facial muscles as a …

We have developed an in situ technique to label individual euchromatic chromosome arms in interspecific crosses between Mus musculus (MMU) and M. spretus (MSP). The MMU and MSP genomes diverged 2-3 million years ago and show an overall sequence divergence of approximately 1 %. Comparative hybridization of MMU versus MSP DNA and subsequent spectral analysis of the euchromatic hybridization profiles discriminated between maternal (MMU) and paternal (MSP) chromosomes in F-1 hybrids. Dispersed repetitive DNA elements were the preferred hybridization target of MMU DNA on maternal chromosomes and of MSP DNA on paternal chromosomes. Differences in centromeric satellite DNAs were detected by conventional fluorescence in situ hybridization and served as internal controls. Our experiments suggest that it is possible, in principle, to discriminate between paternal and maternal chromosomes on the basis of sequence differences.