The relationship between Rose Anna Shedlock (c1850-1878) and Emile Roux (1853-1933).

The famous French scientist, Emile Roux, was previously discovered to have been secretly married to an English woman, Rose Anna Shedlock, one of the first women medical school students in Britain and Europe. Emile and Rose most likely met while in medical school in Paris, although for very different reasons, neither graduated. It was previously suggested that Rose left medical school after only a few years, although we present new evidence that that she was still a medical student four years later when she would have been near completion. Regardless, Rose moved back to England prior to taking her qualifying exams, where we found she lived at a girl's boarding school where one of her sisters was head mistress. In the following year, Emile travelled to London where he and Rose were married in a quiet civil ceremony. Soon after the wedding, Emile returned to Paris where he began working as an assistant to Louis Pasteur. In a tragic twist of fate, Rose died a year later in Madeira, which we have now noted was within days of when Emile performed his breakthrough experiments that led to the creation of vaccines in the laboratory.

High-throughput screen to identify compounds that prevent or target telomere loss in human cancer cells.

Chromosome instability (CIN) is an early step in carcinogenesis that promotes tumor cell progression and resistance to therapy. Using plasmids integrated adjacent to telomeres, we have previously demonstrated that the sensitivity of subtelomeric regions to DNA double-strand breaks (DSBs) contributes to telomere loss and CIN in cancer. A high-throughput screen was created to identify compounds that affect telomere loss due to subtelomeric DSBs introduced by I-SceI endonuclease, as detected by cells expressing green fluorescent protein (GFP). A screen of a library of 1832 biologically-active compounds identified a variety of compounds that increase or decrease the number of GFP-positive cells following activation of I-SceI. A curated screen done in triplicate at various concentrations found that inhibition of classical nonhomologous end joining (C-NHEJ) increased DSB-induced telomere loss, demonstrating that C-NHEJ is functional in subtelomeric regions. Compounds that decreased DSB-induced telomere loss included inhibitors of mTOR, p38 and tankyrase, consistent with our earlier hypothesis that the sensitivity of subtelomeric regions to DSBs is a result of inappropriate resection during repair. Although this assay was also designed to identify compounds that selectively target cells experiencing telomere loss and/or chromosome instability, no compounds of this type were identified in the current screen.

Telomeric Double Strand Breaks in G1 Human Cells Facilitate Formation of 5' C-Rich Overhangs and Recruitment of TERRA.

Telomeres, repetitive nucleoprotein complexes that protect chromosomal termini and prevent them from activating inappropriate DNA damage responses (DDRs), shorten with cell division and thus with aging. Here, we characterized the human cellular response to targeted telomeric double-strand breaks (DSBs) in telomerase-positive and telomerase-independent alternative lengthening of telomere (ALT) cells, specifically in G1 phase. Telomeric DSBs in human G1 cells elicited early signatures of a DDR; however, localization of 53BP1, an important regulator of resection at broken ends, was not observed at telomeric break sites. Consistent with this finding and previously reported repression of classical non-homologous end-joining (c-NHEJ) at telomeres, evidence for c-NHEJ was also lacking. Likewise, no evidence of homologous recombination (HR)-dependent repair of telomeric DSBs in G1 was observed. Rather, and supportive of rapid truncation events, telomeric DSBs in G1 human cells facilitated formation of extensive tracks of resected 5' C-rich telomeric single-stranded (ss)DNA, a previously proposed marker of the recombination-dependent ALT pathway. Indeed, induction of telomeric DSBs in human ALT cells resulted in significant increases in 5' C-rich (ss)telomeric DNA in G1, which rather than RPA, was bound by the complementary telomeric RNA, TERRA, presumably to protect these exposed ends so that they persist into S/G2 for telomerase-mediated or HR-dependent elongation, while also circumventing conventional repair pathways. Results demonstrate the remarkable adaptability of telomeres, and thus they have important implications for persistent telomeric DNA damage in normal human G1/G0 cells (e.g., lymphocytes), as well as for therapeutically relevant targets to improve treatment of ALT-positive tumors.

Defining the mutation signatures of DNA polymerase θ in cancer genomes.

DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of BRCA-mutated cancer cells. It is crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the POLQ-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that BRCA-mutated cancers with a higher level of POLQ expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions of POLQ, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.

The DNA damage response at dysfunctional telomeres, and at interstitial and subtelomeric DNA double-strand breaks.

In mammals, DNA double-strand breaks (DSBs) are primarily repaired by classical non-homologous end joining (C-NHEJ), although homologous recombination repair and alternative NHEJ (A-NHEJ), which involve DSB processing, can also occur. These pathways are tightly regulated to maintain chromosome integrity. The ends of chromosomes, called telomeres, contain telomeric DNA that forms a cap structure in cooperation with telomeric proteins to prevent the activation of the DNA damage response and chromosome fusion at chromosome termini. Telomeres and subtelomeric regions are poor substrates for DNA replication; therefore, regions near telomeres are prone to replication fork stalling and chromosome breakage. Moreover, DSBs near telomeres are poorly repaired. As a result, when DSBs occur near telomeres in normal cells, the cells stop proliferating, while in cancer cells, subtelomeric DSBs induce rearrangements due to the absence of cell cycle checkpoints. The sensitivity of subtelomeric regions to DSBs is due to the improper regulation of processing, because although C-NHEJ is functional at subtelomeric DSBs, excessive processing results in an increased frequency of large deletions and chromosome rearrangements involving A-NHEJ.

Differences in the recruitment of DNA repair proteins at subtelomeric and interstitial I-SceI endonuclease-induced DNA double-strand breaks.

Telomeres are nucleoprotein structures that are required to protect chromosome ends. Dysfunctional telomeres are recognized as DNA double-strand breaks (DSBs), and elicit the activation of a DNA damage response (DDR). We have previously reported that DSBs near telomeres are poorly repaired, resulting in a high frequency of large deletions and gross chromosome rearrangements (GCRs). Our previous genetic studies have demonstrated that this sensitivity of telomeric regions to DSBs is a result of excessive processing. In the current study, we have further investigated the sensitivity of telomeric regions to DSBs through the analysis of repair proteins associated with DSBs at interstitial and telomeric sites. Following the inducible expression of I-SceI endonuclease, chromatin immunoprecipitation (ChIP) and real-time quantitative PCR were used to compare the recruitment of repair proteins at I-SceI-induced DSBs at interstitial and subtelomeric sites. We observed that proteins that are specifically associated with processing of DSBs during homologous recombination repair, RAD51, BRCA1, and CtIP, are present at a much greater abundance at subtelomeric DSBs. In contrast, Ku70, which is specifically involved in classical nonhomologous end joining, showed no difference at interstitial and subtelomeric DSBs. Importantly, ATM was lower in abundance at subtelomeric DSBs, while ATR was in greater abundance at subtelomeric DSBs, consistent with the accumulation of processed DSBs near telomeres, since processing is accompanied by a transition from ATM to ATR binding. Combined, our results suggest that excessive processing is responsible for the increased frequency of large deletions and GCRs at DSBs near telomeres.

Replication Stress and Telomere Dysfunction Are Present in Cultured Human Embryonic Stem Cells.

Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability, which can result in replicative senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans, making it important to minimize the levels of replication stress and telomere dysfunction. Here, the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A, exogenous nucleosides, or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast, adding exogenous nucleosides relieved dysfunction, suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However, because the loss of telomeres can lead to chromosome instability and cancer, we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction.

Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks.

The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3'-5' exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.

Numerical chromosomal instability mediates susceptibility to radiation treatment.

The exquisite sensitivity of mitotic cancer cells to ionizing radiation (IR) underlies an important rationale for the widely used fractionated radiation therapy. However, the mechanism for this cell cycle-dependent vulnerability is unknown. Here we show that treatment with IR leads to mitotic chromosome segregation errors in vivo and long-lasting aneuploidy in tumour-derived cell lines. These mitotic errors generate an abundance of micronuclei that predispose chromosomes to subsequent catastrophic pulverization thereby independently amplifying radiation-induced genome damage. Experimentally suppressing whole-chromosome missegregation reduces downstream chromosomal defects and significantly increases the viability of irradiated mitotic cells. Further, orthotopically transplanted human glioblastoma tumours in which chromosome missegregation rates have been reduced are rendered markedly more resistant to IR, exhibiting diminished markers of cell death in response to treatment. This work identifies a novel mitotic pathway for radiation-induced genome damage, which occurs outside of the primary nucleus and augments chromosomal breaks. This relationship between radiation treatment and whole-chromosome missegregation can be exploited to modulate therapeutic response in a clinically relevant manner.

Effect of radiation quality on mutagenic joining of enzymatically-induced DNA double-strand breaks in previously irradiated human cells.

Previous work has shown that high charge and energy particle irradiation of human cells evokes a mutagenic repair phenotype, defined by increased mutagenic repair of new double-strand breaks that are introduced enzymatically, days or weeks after the initial irradiation. The effect was seen originally with 600 MeV/u (56)Fe particles, which have a linear energy transfer (LET) value of 174 keV/µm, but not with X rays or γ rays (LET ≤ 2 keV/µm). To better define the radiation quality dependence of the phenomenon, we tested two ions with intermediate LET values, 1,000 MeV/u (48)Ti (LET = 108 keV/µm) and 300 MeV/u (28)Si (LET = 69 keV/µm). These experiments used a previously validated assay, where a rare-cutting nuclease introduces double-strand breaks in two reporter transgene cassettes, which are located on different chromosomes. Deletions of a block of sequence in one of the cassettes, or translocations between cassettes, are measured independently using a multicolor fluorescence assay. The results showed that (48)Ti was a potent, but transient, inducer of mutagenic repair, based on increased frequency of nuclease-induced translocations. The (48)Ti ions did not affect the frequency of nuclease-induced deletions. The (28)Si ions had no measurable effect on either endpoint. There was a close correlation between the induction of the mutagenic repair phenomenon and the frequency of micronuclei in the targeted population (R(2) = 0.74), whereas there was no apparent correlation with radiation-induced cell inactivation. Together, these results better define the radiation quality dependence of the mutagenic repair phenomenon and establish its correlation, or lack of correlation, with other endpoints.

DNA-damage response during mitosis induces whole-chromosome missegregation.

UNLABELLED: Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. SIGNIFICANCE: The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities.

Increased mutagenic joining of enzymatically-induced DNA double-strand breaks in high-charge and energy particle irradiated human cells.

The carcinogenic risk of high-charge and energy (HZE) particle exposure arises from its ability to both induce complex DNA damage and from its ability to evoke deleterious, non-DNA targeted effects. We investigate here whether these nontargeted effects involve dysregulation of double-strand break repair, such that a history of HZE exposure heightens the risks from future injury. We used a new human cell reporter line, in which expression of the I-SceI meganuclease stimulates both translocations on different chromosomes, and deletions on the same chromosome. Exposure to 1.0 Gy of 600 MeV/u (56)Fe ions led to a doubling in the frequency of I-SceI-mediated translocations and a smaller, but nevertheless significant, increase in the frequency of I-SceI-mediated deletions. This mutagenic repair phenotype persisted for up to two weeks and eight population doublings. The phenotype was not induced by low-linear energy transfer radiation or by a lower dose of HZE-particle radiation (0.3 Gy) indicating that the effect is radiation quality and dose dependent. The mutagenic repair phenotype was associated with the presence of micronuclei and persistent DSB repair foci, consistent with a hypothesis that genomic stress is a causative factor.

The role of ATM in the deficiency in nonhomologous end-joining near telomeres in a human cancer cell line.

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.

Mechanisms of telomere loss and their consequences for chromosome instability.

The ends of chromosomes in mammals, called telomeres, are composed of a 6-bp repeat sequence, TTAGGG, which is added on by the enzyme telomerase. In combination with a protein complex called shelterin, these telomeric repeat sequences form a cap that protects the ends of chromosomes. Due to insufficient telomerase expression, telomeres shorten gradually with each cell division in human somatic cells, which limits the number of times they can divide. The extensive cell division involved in cancer cell progression therefore requires that cancer cells must acquire the ability to maintain telomeres, either through expression of telomerase, or through an alternative mechanism involving recombination. It is commonly thought that the source of many chromosome rearrangements in cancer cells is a result of the extensive telomere shortening that occurs prior to the expression of telomerase. However, despite the expression of telomerase, tumor cells can continue to show chromosome instability due to telomere loss. Dysfunctional telomeres in cancer cells can result from oncogene-induced replication stress, which results in double-strand breaks (DSBs) at fragile sites, including telomeres. DSBs near telomeres are especially prone to chromosome rearrangements, because telomeric regions are deficient in DSB repair. The deficiency in DSB repair near telomeres is also an important mechanism for ionizing radiation-induced replicative senescence in normal human cells. In addition, DSBs near telomeres can result in chromosome instability in mouse embryonic stem cells, suggesting that telomere loss can contribute to heritable chromosome rearrangements. Consistent with this possibility, telomeric regions in humans are highly heterogeneous, and chromosome rearrangements near telomeres are commonly involved in human genetic disease. Understanding the mechanisms of telomere loss will therefore provide important insights into both human cancer and genetic disease.

Telomere dysfunction and chromosome instability.

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair.

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.

Telomere loss as a mechanism for chromosome instability in human cancer.

Cancer cells commonly have a high rate of telomere loss, even when expressing telomerase, contributing to chromosome instability and tumor cell progression. This review addresses the hypothesis that this high rate of telomere loss results from a combination of four factors. The first factor is an increase in the frequency of double-strand breaks (DSB) at fragile sites in cancer cells due to replication stress. The second factor is that telomeres are fragile sites. The third factor is that subtelomeric regions are highly sensitive to DSBs, so that DSBs near telomeres have an increased probability of resulting in chromosome instability. The fourth factor is that cancer cells may be deficient in chromosome healing, the de novo addition of telomeres to the sites of DSBs, a mechanism that prevents chromosome instability resulting from DSBs near telomeres. Understanding these factors and how they influence telomere loss will provide important insights into the mechanisms of chromosome instability and the development of novel approaches for anti-cancer therapy. Cancer Res; 70(11); 4255-9. (c)2010 AACR.

Effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to DNA double-strand breaks in a human tumor cell line.

The ends of chromosomes, called telomeres, are composed of a DNA repeat sequence and associated proteins, which prevent DNA degradation and chromosome fusion. We have previously used plasmid sequences integrated adjacent to a telomere to demonstrate that mammalian telomeres suppress gene expression, called telomere position effect (TPE). We have also shown that subtelomeric regions are highly sensitive to double-strand breaks, leading to chromosome instability, and that this instability can be prevented by the addition of a new telomere to the break, a process called chromosome healing. We have now targeted the same plasmid sequences to a site 100 kb from a telomere in a human carcinoma cell line to address the effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to double-strand breaks. The results demonstrate a substantial decrease in TPE 100 kb from the telomere, demonstrating that TPE is very limited in range. Chromosome healing was also diminished 100 kb from the telomere, consistent with our model that chromosome healing serves as a repair process for restoring lost telomeres. Conversely, the region 100 kb from the telomere was highly sensitive to double-strand breaks, demonstrating that the sensitive region is a relatively large target for ionizing radiation-induced chromosome instability.