Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres.

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise approximately 17% of human DNA. The average human genome contains approximately 80-100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (EN(i)) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair. Here we have characterized EN(i) retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, approximately 30% of EN(i) retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among EN(i) retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated EN(i) retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of EN(i) retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends. Thus, we propose that EN(i) retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.

DNA repair mediated by endonuclease-independent LINE-1 retrotransposition.

Long interspersed elements (LINE-1s) are abundant retrotransposons in mammalian genomes that probably retrotranspose by target site-primed reverse transcription (TPRT). During TPRT, the LINE-1 endonuclease cleaves genomic DNA, freeing a 3' hydroxyl that serves as a primer for reverse transcription of LINE-1 RNA by LINE-1 reverse transcriptase. The nascent LINE-1 cDNA joins to genomic DNA, generating LINE-1 structural hallmarks such as frequent 5' truncations, a 3' poly(A)+ tail and variable-length target site duplications (TSDs). Here we describe a pathway for LINE-1 retrotransposition in Chinese hamster ovary (CHO) cells that acts independently of endonuclease but is dependent upon reverse transcriptase. We show that endonuclease-independent LINE-1 retrotransposition occurs at near-wildtype levels in two mutant cell lines that are deficient in nonhomologous end-joining (NHEJ). Analysis of the pre- and post-integration sites revealed that endonuclease-independent retrotransposition results in unusual structures because the LINE-1s integrate at atypical target sequences, are truncated predominantly at their 3' ends and lack TSDs. Moreover, two of nine endonuclease-independent retrotranspositions contained cDNA fragments at their 3' ends that are probably derived from the reverse transcription of endogenous mRNA. Thus, our results suggest that LINE-1s can integrate into DNA lesions, resulting in retrotransposon-mediated DNA repair in mammalian cells.

Impact of telomerase ablation on organismal viability, aging, and tumorigenesis in mice lacking the DNA repair proteins PARP-1, Ku86, or DNA-PKcs.

The DNA repair proteins poly(ADP-ribose) polymerase-1 (PARP-1), Ku86, and catalytic subunit of DNA-PK (DNA-PKcs) have been involved in telomere metabolism. To genetically dissect the impact of these activities on telomere function, as well as organismal cancer and aging, we have generated mice doubly deficient for both telomerase and any of the mentioned DNA repair proteins, PARP-1, Ku86, or DNA-PKcs. First, we show that abrogation of PARP-1 in the absence of telomerase does not affect the rate of telomere shortening, telomere capping, or organismal viability compared with single telomerase-deficient controls. Thus, PARP-1 does not have a major role in telomere metabolism, not even in the context of telomerase deficiency. In contrast, mice doubly deficient for telomerase and either Ku86 or DNA-PKcs manifest accelerated loss of organismal viability compared with single telomerase-deficient mice. Interestingly, this loss of organismal viability correlates with proliferative defects and age-related pathologies, but not with increased incidence of cancer. These results support the notion that absence of telomerase and short telomeres in combination with DNA repair deficiencies accelerate the aging process without impacting on tumorigenesis.

Role of mammalian Rad54 in telomere length maintenance.

The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.

Postreplicative joining of DNA double-strand breaks causes genomic instability in DNA-PKcs-deficient mouse embryonic fibroblasts.

Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNA-PKcs-/- MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs-/- cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs-/- MEFs than in their wild-type counterparts. Spectral karyotyping-fluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs-/- MEFs. Because DNA-PKcs-/- MEFs show a strong G1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs-/- MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events.

Novel localization of the DNA-PK complex in lipid rafts: a putative role in the signal transduction pathway of the ionizing radiation response.

Increased sensitivity to ionizing radiation (IR) has been shown to be due to defects in DNA double-strand break repair machinery. The major pathway in mammalian cells dedicated to the repair of DNA double-strand breaks is by the nonhomologous end-joining machinery. Six components function in this pathway, of which three (Ku70, Ku86, and DNA-PKcs) constitute a protein complex known as DNA-dependent protein kinase (DNA-PK). However, it is now recognized that the cellular radiation response is complex, and radiosensitivity may be also regulated at different levels in the radiation signal transduction pathway. In addition to DNA damage, exposure to IR triggers intracellular signaling cascades that overlap with pathways initiated by ligand engagement to a receptor. In this study, we provide evidence for the novel localization of the DNA-PK complex in lipid rafts. We also show this property is not a generalized characteristic of all DNA repair proteins. Furthermore, we have detected Ku86 in yeast lipid rafts. Our results suggest that the components of this complex might be recruited separately to the plasma membrane by tethering with raft-resident proteins. In addition, we found an irradiation-induced differential protein phosphorylation pattern dependent upon DNA-PKcs in lipid rafts. Thus, we speculate that another role for the DNA-PKcs subunit and perhaps for the holoenzyme is in the signal transduction of IR response.

Functional interaction between DNA-PKcs and telomerase in telomere length maintenance.

DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex that functions in the non-homologous end-joining of double-strand breaks, and it has been shown previously to have a role in telomere capping. In particular, DNA-PKcs deficiency leads to chromosome fusions involving telomeres produced by leading-strand synthesis. Here, by generating mice doubly deficient in DNA-PKcs and telomerase (Terc(-/-)/DNA-PKcs(-/-)), we demonstrate that DNA-PKcs also has a fundamental role in telomere length maintenance. In particular, Terc(-/-)/DNA-PKcs(-/-) mice displayed an accelerated rate of telomere shortening when compared with Terc(-/-) controls, suggesting a functional interaction between both activities in maintaining telomere length. In addition, we also provide direct demonstration that DNA-PKcs is essential for both end-to-end fusions and apoptosis triggered by critically short telomeres. Our data predict that, in telomerase-deficient cells, i.e. human somatic cells, DNA-PKcs abrogation may lead to a faster rate of telomere degradation and cell cycle arrest in the absence of increased apoptosis and/or fusion of telomere-exhausted chromosomes. These results suggest a critical role of DNA-PKcs in both cancer and aging.