Unlimited cellular proliferation depends on counteracting the telomere attrition that accompanies DNA replication. In human cancers this usually occurs through upregulation of telomerase activity, but in 10-15% of cancers - including some with particularly poor outcome - it is achieved through a mechanism known as alternative lengthening of telomeres (ALT). ALT, which is dependent on homologous recombination, is therefore an important target for cancer therapy. Although dissection of the mechanism or mechanisms of ALT has been challenging, recent advances have led to the identification of several genes that are required for ALT and the elucidation of the biological significance of some phenotypic markers of ALT. This has enabled development of a rapid assay of ALT activity levels and the construction of molecular models of ALT.

Prior evidence has supported the existence of multiple susceptibility genes for schizophrenia. Multipoint linkage analysis of the 270 Irish high-density pedigrees that we have studied, as well as results from several other samples, suggest that at least one such gene is located in region 6p24-21. In the present study, family-based association analysis of 36 simple sequence-length-polymorphism markers and of 17 SNP markers implicated two regions, separated by approximately 7 Mb. The first region, and the focus of this report, is 6p22.3. In this region, single-nucleotide polymorphisms within the 140-kb gene DTNBP1 (dystrobrevin-binding protein 1, or dysbindin) are strongly associated with schizophrenia. Uncorrected, empirical P values produced by the program TRANSMIT were significant (P<.01) for a number of individual SNP markers, and most remained significant when the data were restricted to include only one affected offspring per nuclear family per extended pedigree; multiple three-marker haplotypes were highly significant (P=.008-.0001) under the restricted conditions. The pattern of linkage disequilibrium is consistent with the presence of more than one susceptibility allele, but this important issue is unresolved. The number of markers tested in the adjacent genes, all of which are negative, is not sufficient to rule out the possibility that the dysbindin gene is not the actual susceptibility gene, but this possibility appears to be very unlikely. We conclude that further investigation of dysbindin is warranted.

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.Telomeres are the protein-DNA structures at chromosome termini in eukaryotic cells that protect chromosomes from end-to-end fusion and likely function as a trigger for proliferative arrest in mammalian cells. In mammals, telomeric DNA consists of a tandem array of the 6-nucleotide (nt) repeat 5ЈTTAGGG3Ј/3ЈCCCTAA5Ј (26), approximately 4 to 14 kb long in humans (10), terminating in a 150-to 200-nt 3Ј singlestrand DNA overhang of the G-rich strand (23,46). A structural solution to chromosome end protection is provided by sequestering the 3Ј telomeric overhang in a displacement loop within the intratelomeric duplex DNA. We identified this structure, termed a telomere loop (t-loop), following electron microscopic (EM) examination of telomeric DNA isolated from in situ photo-cross-linked genomic DNA from human and mouse nuclei (13). t-loops have also been observed in plant (7), avian (31), and multiple protist species (27, 28), suggesting that t-loops are a common mechanism for chromosome protection among eukaryotes.Due to the end replication problem (33, 45), oxidative damage (44), and possible nucleolytic processing (23), telomeres in human cells erode by approximately 100 bp with each cell division (14). In human somatic tissue, little or no telomerase activity is present, resulting in telomere-induced cellular senescence after many population doublings (14,15,24). If senescence is bypassed by mutations or expression of viral oncogenes, the cell will continue to divide until crisis and eventual cell death (12). However, human cells can stave off induction of senescence or escape crisis by regenerating the telomeric DNA (4,9,17). This occurs most commonly, in approximately 90 t...

Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader͞DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC͞9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA. E ukaryotic cells exposed to genotoxic agents activate the DNA damage checkpoint signaling pathway, which arrests cellcycle progression and in so doing prevents cell death or mutations. Recent work has revealed that in mammalian cells, the ATM and ATR proteins, which belong to the phosphatidylinositide kinase-like kinase family, and the Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complexes, which have structural similarities to the replication clamp loader and replication clamp RFC and proliferating cell nuclear antigen (PCNA), respectively, are involved in damage recognition, which activates the checkpoint response (reviewed in refs. 1-4). Studies with budding and fission yeasts have shown that the orthologs of these proteins perform similar functions. However, biochemical data on the specific roles of the phosphatidylinositide kinase-like kinase family members and the Rad17-RFC and 9-1-1 complexes are scarce, and hence the damage sensing step of the checkpoint response remains illdefined. We previously reported that ATR directly recognizes and is activated by damaged DNA (5). In this article, we investigate the interactions of Rad17-RFC and the 9-1-1 checkpoint complexes with DNA to gain some insight into their roles as damage sensors.Rad17-RFC is one of the three known RFC-like complexes in mammalian cells. In this form of RFC, the p140 subunit is replaced by the 75-kDa Rad17 protein, which has homology to all RFC subunits (6). Yeast genetic studies indicate that the orthologs of human Rad17 function exclusively in the DNA damage checkpoint response (7,8). The 9-1-1 checkpoint complex is a heterotrimer of Rad9, Rad1, and Hus1 proteins, which were predicted to have structural homology to PCNA (9-13). Previously, we showed that Rad17 associates with the four small RFC subunits to make an RFC-like complex, which by electron microscopy exhibits an RFC-like structure (14). Similarly, we found that Rad9, Rad1, and Hus1 f...

Summary Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres in human cells. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome cohesion resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 (Telomere Repeat binding Factor 2) from telomeres, telomeric 3’-overhang degradation and ATM (Ataxia Telangiectasia Mutated) activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration monitoring system that reacts to improper progression through mitosis.

Five dysfunctional telomeres predict onset of senescence in human cellsReplicative senescence is triggered by DNA damage response foci associated with telomeres. Reddel and colleagues now establish that a threshold of five damaged telomeres exists to induce senescence in normal cells and that end-to-end chromosome fusion is not required for senescence induction.

Summary Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion resistant intermediate-state activate differential ATM signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur.