Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3\u27 overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway

Dysfunctional telomeres activate an ATM-ATR-dependent DNA damage response to suppress tumorigenesis

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM-dependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo

POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination

POT1 (protection of telomere 1) is a highly conserved single-stranded telomeric binding protein that is essential for telomere end protection. Here, we report the cloning and characterization of a second member of the mouse POT family. POT1b binds telomeric DNA via conserved DNA binding oligonucleotide/oligosaccharide (OB) folds. Compared to POT1a, POT1b OB-folds possess less sequence specificity for telomeres. In contrast to POT1a, truncated POT1b possessing only the OB-folds can efficiently localize to telomeres in vivo. Overexpression of a mutant Pot1b allele that cannot bind telomeric DNA initiated a DNA damage response at telomeres that led to p53-dependent senescence. Furthermore, a reduction of the 3′ G-rich overhang, increased chromosomal fusions and elevated homologous recombination (HR) were observed at telomeres. shRNA mediated depletion of endogenous Pot1b in Pot1a deficient cells resulted in increased chromosomal aberrations. Our results indicate that POT1b plays important protective functions at telomeres and that proper maintenance of chromosomal stability requires both POT proteins