Telomere elongation involves intra-molecular DNA replication in cells utilizing alternative lengthening of telomeres

Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2′ DNA and the resulting protein–DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn(2+)-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms

TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3′ overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks, but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after γ-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates

Quantitative theory of telomere length regulation and cellular senescence

In normal somatic cells, telomere length shortens with each cell replication. This progressive shortening is associated with cellular senescence and apoptosis. Germ cells, stem cells, and the majority of cancer cells express telomerase, an enzyme that extends telomere length and, when expressed at sufficient levels, can immortalize or extend the life span of a cell line. It is believed that telomeres switch between two states: capped and uncapped. The telomere state determines its accessibility to telomerase and also the onset of senescence. One hypothesis is that the t loop, a large lariat-like structure, represents the capped state. In this paper we model a telomere state on the basis of the biophysics of t-loop formation, allowing us to develop a single mathematical model that accounts for two processes: telomere length regulation for telomerase positive cells and cellular senescence in somatic cells. The model predicts the steady-state length distribution for telomerase positive cells, describes the time evolution of telomere length, and computes the life span of a cell line on the basis of the levels of TRF2 and telomerase expression. The model reproduces a wide range of experimental behavior and fits data from immortal cell lines (HeLa S3 and 293T) and somatic cells (human diploid fibroblasts) well. We conclude that the t loop as the capped state is a quantitatively reasonable model of telomere length regulation and cellular senescence

Topical DNA oligonucleotide therapy reduces UV-induced mutations and photocarcinogenesis in hairless mice

UV-induced DNA damage gives rise to mutations and skin cancer. We have previously reported that treatment of skin cells in vitro with thymidine dinucleotide (pTT) activates p53 and increases the ability of cells to repair subsequent UV-induced DNA damage by enhancing endogenous DNA repair capacity. Here we show that topical pTT pretreatment enhances the rate of DNA photoproduct removal, decreases UV-induced mutations, and reduces photocarcinogenesis in UV-irradiated hairless WT repair-proficient and Xpc(+/-) heterozygous partially repair-deficient mice, both transgenic for the lacZ/pUR288 mutation-indicator gene. These data support the existence of inducible mammalian DNA damage responses that increase DNA repair capacity after DNA damage and hence reduce the impact of future exposures to environmental carcinogens. The ability of topically applied pTT to induce protective physiologic responses that normally result from DNA damage suggests a previously undescribed means of reducing skin cancer in high-risk individuals