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Fluorescence in situ Hybridization (FISH) techniques, including whole chromosome painting (WCP), spectral karyotyping (SKY), and multicolor FISH (mFISH), are used extensively to characterize and enumerate inter-chromosomal rearrangements (e.g., translocations). Directional genomic hybridization (dGH) is a relatively new cytogenomics-based methodology that combines the strand-specific strategy of Chromosome Orientation-FISH (CO-FISH) with bioinformatics-driven design of single-stranded DNA probe sets that are unique and of like orientation. Such a strategy produces directional probe sets that hybridize to one-and only one-chromatid of prepared (single-stranded) metaphase chromosomes, thereby facilitating high-resolution visualization of intra-chromosomal rearrangements, specifically inversions, and greatly improving our ability to detect such otherwise cryptic structural variants within the genome. In addition to its usefulness in the study of various disease states, including cancer, relevant applications of dGH include monitoring cytogenetic damage caused by exposure to clastogenic agents (e.g., ionizing radiation). dGH can be applied as a discovery tool to globally assess the integrity of the genome, but it can also be used in a more targeted fashion to interrogate fine structural changes at the kilobase level. Consequently, dGH is capable of providing significant mechanistic insight and information not easily obtainable by other approaches.

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The cytogenomics-based methodology of Directional Genomic Hybridization (dGH™) emerged from the concept of strand-specific hybridization, first made possible by Chromosome Orientation FISH (CO-FISH), the utility of which was demonstrated in a variety of early applications, often involving telomeres. Similar to standard whole chromosome painting (FISH), dGH™ is capable of identifying inter-chromosomal rearrangements (translocations between chromosomes), but its distinctive strength stems from its ability to detect intra-chromosomal rearrangements (inversions within chromosomes), and to do so at higher resolution than previously possible. dGH™ brings together the strand specificity and directionality of CO-FISH with sophisticated bioinformatics-based oligonucleotide probe design to unique sequences. dGH™ serves not only as a powerful discovery tool-capable of interrogating the entire genome at the megabase level-it can also be used for high-resolution targeted detection of known inversions, a valuable attribute in both research and clinical settings. Detection of chromosomal inversions, particularly small ones, poses a formidable challenge for more traditional cytogenetic approaches, especially when they occur near the ends or telomeric regions. Here, we describe Telo-dGH™, a strand-specific scheme that utilizes dGH™ in combination with telomere CO-FISH to differentiate between terminal exchange events, specifically terminal inversions, and an altogether different form of genetic recombination that often occurs near the telomere, namely sister chromatid exchange (SCE).

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Chromosome ends are shielded from exonucleolytic attack and inappropriate end-joining by terminal structures called telomeres; these structures are potential targets for anticancer drugs. Telomeres are composed of a simple DNA sequence (5?-TTAGGG-3? in humans) repeated more than a thousand times, a short 3? single-stranded overhang, and numerous proteins. Electron microscopy has shown that the 3? overhang pairs with the complementary strand at an internal site creating a small displacement loop and a large double-stranded "t-loop." Our goal is to determine whether all telomeres adopt the t-loop configuration, or whether there are two or more distinct configurations. Progress in optimizing super-resolution (SR) microscopy for this ongoing investigation is reported here. Results suggest that under certain conditions sample preparation procedures may disrupt chromatin by causing loss of nucleosomes. This finding may limit the use of SR microscopy in telomere studies.

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Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1-2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.

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Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions-reversals in the orientation of DNA sequences within a chromosome-should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting-a whole new way of looking at structural features of the genome-that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility.

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Fluorescence in situ hybridization (FISH) has become a powerful tool for exploring genomes at the level of chromosomes. The procedure can be used to identify individual chromosomes, rearrangements between chromosomes, and the location within a chromosome of specific DNA sequences such as centromeres, telomeres, and even individual genes. Chromosome orientation FISH (CO-FISH) extends the information obtainable from standard FISH to include the relative orientation of two or more DNA sequences within a chromosome (Goodwin and Meyne, Cytogenet Cell Genet 63:126-127, 1993). In combination with a suitable reference probe, CO-FISH can also determine the absolute 5'-3' direction of a DNA sequence relative to the short arm (pter) to long arm (qter) axis of the chromosome. This variation of CO-FISH was originally termed "COD-FISH" (Chromosome orientation and direction FISH) to reflect this fact (Meyne and Goodwin, Chromosome Research 3:375-378, 1995). Telomeric DNA serves as a convenient and absolute reference probe for this purpose, since all G-rich 5'-(TTAGGG)( n )-3' telomeric sequences are terminally located and oriented away from the centromere.In the beginning, CO-FISH was used to detect obligate chromosomal inversions associated with isochromosome formation (Bailey et al., Mutagenesis 11:139-144, 1996), various pericentric inversions (Bailey et al., Cytogenetics and Cell Genetics 75:248-253, 1996), and to confirm the origin of centromeric lateral asymmetry (Goodwin et al., Chromosoma 104:345-347, 1996). More recent and sophisticated applications of CO-FISH include distinction between telomeres produced via leading- vs. lagging-strand DNA synthesis (Bailey et al., Science 293:2462-2465, 2001), identification of interstitial blocks of telomere sequence that result from inappropriate fusion to double-strand breaks (telomere-DSB fusion) (Bailey et al., DNA Repair (Amst) 3:349-357, 2004), discovery of elevated rates of mitotic recombination at chromosomal termini (Cornforth and Eberle, Mutagenesis, 16:85-89, 2001) and sister chromatid exchange within telomeric DNA (T-SCE) (Bailey et al., Nucleic Acids Res 32:3743-3751, 2004), establishing replication timing of mammalian telomeres throughout S-phase (ReD-FISH) (Cornforth et al., In: Cold Spring Harbor Symposium: Telomeres and Telomerase, Cold Spring Harbor, NY, 2003; Zou et al., Proc Natl Acad Sci USA 101:12928-12933, 2004) and in combination with -spectral karyotyping (SKY-CO-FISH) (Williams et al., Cancer Res 69:2100-2107, 2009). For more information, the reader is referred to several reviews (Bailey et al., Cytogenet Genome Res 107, 14-17, 2004; Bailey and Cornforth, Cell Mol Life Sci 64:2956-2964, 2007; Bailey, Telomeres and Double-Strand Breaks - All's Well that "Ends" Well, Radiat Res 169:1-7, 2008).

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Telomeres are a hotspot for sister chromatid exchange (T-SCE). Any biological consequence of this form of instability remained obscure until quantitative modeling revealed a link between elevated T-SCE rates and accelerated cellular replicative senescence. This work strongly suggests that progressive telomere erosion is not the only determinant of replicative capacity; instead, T-SCE need to be considered as an independent factor controlling colony growth and senescence. Additionally high T-SCE rates have been observed in cells with deficiencies in WRN and BLM, the genes that are defective in Werner's and Bloom's syndromes, implying a connection to premature aging. In this Research Perspective we will explore some of the implications this recent work has for human health.

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Chromosome Orientation FISH (CO-FISH) is a technique that can be used to extend the information obtainable from standard FISH to include the relative orientation of two or more DNA sequences within a chromosome. CO-FISH can determine the absolute 5'-to-3' direction of a DNA sequence relative to the short arm-to-long arm axis of the chromosome, and so was originally termed "COD-FISH" (Chromosome Orientation and Direction FISH). CO-FISH has been employed to detect chromosomal inversions associated with isochromosome formation, various pericentric inversions, and to confirm the origin of lateral asymmetry. More recent and sophisticated applications of CO-FISH include distinction between telomeres produced via leading- vs. lagging-strand DNA synthesis, identification of interstitial blocks of telomere sequence that result from inappropriate fusion to double-strand breaks (telomere-DSB fusion), discovery of elevated rates of mitotic recombination at chromosomal termini and sister chromatid exchange within telomeric DNA (T-SCE), establishing replication timing of mammalian telomeres throughout S-phase (ReD-FISH) and to identify chromosomes, in combination with spectral karyotyping (SKY-CO-FISH).

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Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerase-positive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE-but not G-SCE-frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescence may be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome.

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Over the past two decades, our understanding of radiation biology has undergone a fundamental shift in paradigms away from deterministic 'hit-effect' relationships and towards complex ongoing 'cellular responses'. These responses include now familiar, but still poorly understood, phenomena associated with radiation exposure such as genomic instability and bystander effects. Although these responses share some common features (e.g. they occur at high frequency following very low doses, are heterogeneous in their induction and are observed at time points far removed from the initial radiation exposure), the precise relationship between genomic instability and bystander effects remains to be elucidated. This review will provide a synthesis of the known, and proposed, interrelationships among irradiated and bystander cellular responses to radiation. It also discusses our current experimental approach for gaining a clearer understanding of the relationship between damage induction and long-term effects in both irradiated and bystander cells.

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Telomerase-negative cancer cells show increased telomere sister chromatid exchange (T-SCE) rates, a phenomenon that has been associated with an alternative lengthening of telomeres (ALT) mechanism for maintaining telomeres in this subset of cancers. Here we examine whether or not T-SCE can maintain telomeres in human cells using a combinatorial model capable of describing how telomere lengths evolve over time. Our results show that random T-SCE is unlikely to be the mechanism of telomere maintenance of ALT human cells, but that increased T-SCE rates combined with a recently proposed novel mechanism of non-random segregation of chromosomes with long telomeres preferentially into the same daughter cell during cell division can stabilize chromosome ends in ALT cancers. At the end we discuss a possible experiment that can validate the findings of this study.

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Over the past two decades, our understanding of radiation biology has undergone a fundamental shift in paradigms away from deterministic "hit-effect" relationships and towards complex ongoing "cellular responses". These responses include now familiar, but still poorly understood, phenomena associated with radiation exposure such as bystander effects, genomic instability, and adaptive responses. All three have been observed at very low doses, and at time points far removed from the initial radiation exposure, and are extremely relevant for linear extrapolation to low doses; the adaptive response is particularly relevant when exposure is spread over a period of time. These are precisely the circumstances that are most relevant to understanding cancer risk associated with environmental and occupational radiation exposures. This review will provide a synthesis of the known, and proposed, interrelationships amongst low-dose cellular responses to radiation. It also will examine the potential importance of non-targeted cellular responses to ionizing radiation in setting acceptable exposure limits especially to low-LET radiations.

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For cells on the path to carcinogenesis, the key to unlimited growth potential lies in overcoming the steady loss of telomeric sequence commonly referred to as the 'end-replication problem' that occurs with each cell division. Most human tumors have reactivated telomerase, a specialized reverse transcriptase that directs RNA-templated addition of telomeric repeats on to chromosomal termini. However, approximately 10% of tumors maintain their telomeres through a recombination-based mechanism, termed alternative lengthening of telomeres or ALT. Here we demonstrate that telomeric DNA undergoes a high rate of a particular type of recombination visualized cytogenetically as sister chromatid exchange (SCE), and that this rate is dependent on genotype. A novel model of ALT is presented in which it is argued that telomeric exchanges, if they are unequal and occur at a sufficiently high frequency, will allow cells to proliferate indefinitely without polymerase-mediated extension of telomeric sequence.

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The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.

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In addition to joining broken DNA strands, several non-homologous end-joining (NHEJ) proteins have a second seemingly antithetical role in constructing functional telomeres, the nucleoprotein structures at the termini of linear eukaryotic chromosomes that prevent joining between natural chromosome ends. Although NHEJ deficiency impairs double-strand break (DSB) repair, it also promotes inappropriate chromosomal end fusions that are observed microscopically as dicentric chromosomes with telomeric DNA sequence at points of joining. Here, we test the proposition that unprotected telomeres can fuse not only to other dysfunctional telomeres, but also to ends created by DSBs. Severe combined immunodeficiency (scid) is caused by a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PK), an enzyme required for both efficient DSB repair and telomeric end-capping. Cells derived from wild-type, Trp53-/-, scid, and Trp53-/-/scid mice were exposed to gamma radiation to induce DSBs, and chromosomal aberrations were analyzed using a novel cytogenetic technique that can detect joining of a telomere to a DSB end. Telomere-DSB fusions were observed in both cell lines having the scid mutation, but not in wild-type nor Trp53-/- cells. Over a range of 25-340 cGy, half of the visible exchange-type chromosomal aberrations in Trp53-/-/scid cells involved telomere-DSB fusions. Our results demonstrate that unprotected telomeres are not only sensed as, but also acted upon, by the DNA repair machinery as if they were DSB ends. By opening a new pathway for misrepair, telomere-DSB fusion decreases the overall fidelity of DSB repair. The high frequency of these events in scid cells indicates telomere dysfunction makes a strong, and previously unsuspected, contribution to the characteristic radiation sensitivity associated with DNA-PK deficiency.

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The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51.

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As the total dose of X or gamma rays is delivered at lower and lower rates, the yield of chromosome aberrations progressively diminishes. Simultaneously, the shape of the dose response changes from one exhibiting pronounced upward curvature at high dose rates to one approaching linearity at low dose rates. Although the maximum sparing effect caused by lowering the dose rate can be predicted from classical cytogenetic theory, it has yet to be verified experimentally. Here, noncycling normal human fibroblasts were exposed to graded doses of (137)Cs gamma rays at chronic dose rates of 6.3 and 2.8 cGy h(-1), dose rates that we reasoned should be lower than those required to achieve maximal sparing. This was indeed shown to be the case, after it was determined that the two chronic dose rates produced identical linear dose responses of 0.05 total aberrations per cell Gy(-1). Consistent with cytogenetic theory, this value was statistically indistinguishable from the linear coefficient derived from a fit to aberration frequencies produced by high-dose-rate exposure. Exposure to (238)Pu alpha particles also produced a linear dose response for total aberrations, whose slope-with respect to (137)Cs gamma rays as a reference radiation-implied a maximum RBE of 35 +/- 2.

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