Telomeres protect chromosome ends and are critical for maintaining a stable genome. Failure of this protection system results in genome instability that leads to diseases such as cancer and premature aging. Many proteins involved in DNA repair, replication, and recombination are associated with telomeres and mediate the protective function of telomeres. It is therefore important to understand the in vivo roles of DNA metabolic proteins and the consequences of mutations in these proteins at telomeres. Our long-term goal is to understand how the DNA replication factors contribute to the maintenance of telomere integrity. A critical role of the DNA replication machinery in telomerase-mediated telomere maintenance has been suggested from studies of yeast cells. However, how the components of the replication machinery influence telomerase activity and telomere structure is poorly understood in human cancer cells. In this proposal, we will investigate one such factor, human FEN1 protein, a nuclease that is required for normal maturation of Okazaki fragments during DNA replication. Our preliminary studies demonstrate that hFEN1 is involved in generating the single- stranded G-overhangs at telomere ends, and that a deficiency in hFEN1 induces telomere shortening in cancer cells. Hypothesis: the hFEN1 nuclease degrades telomere DNA on the C-rich strand after telomere DNA replication, generating single-stranded G-overhangs. Deficiency in hFEN1 may compromise G-overhang generation and make telomere ends poor substrates for telomerase, leading to disruption of telomerase- mediated telomere maintenance in cancer cells.
Specific aims : 1. To determine whether the nuclease activity of hFEN1 is responsible for G-overhang generation in vivo and in vitro. A dominant-negative nuclease- deficient hFEN1 will be overexpressed in human cells and its effect on telomere structure and telomere length will be analyzed. An in vitro system will be established to examine the ability of hFEN1 protein to process telomere DNA ends. 2. To determine the effect of hFEN1 on leading and lagging daughter telomeres. Leading and lagging daughter telomeres will be separated after one round of DNA replication and the effects of FEN1 deficiency on these two daughter telomeres will be analyzed. 3. To determine the effects of hFEN1 on the recruitment of telomerase to telomeres. A co-immunoprecipitation method will be used to detect physical interaction of hFEN1 with telomerase during the S phase of the cell cycle. The chromatin immunoprecipitation assay will be used to examine any alteration in the recruitment of telomerase to telomeres in FEN1 deficient cells. The findings from these studies will aid us in understanding telomere maintenance mechanism in cancer cells and facilitate the development of new cancer therapy. Relevance to public health: Maintenance of telomere integrity is essential for the unlimited growth of cancer cells. This proposal aims to yield new insights into telomere maintenance in cancer cells, cell growth control and oncogenesis. It may lead to the development of new anti-cancer therapy. ? ? ?
|Gu, Peili; Min, Jin-Na; Wang, Yang et al. (2012) CTC1 deletion results in defective telomere replication, leading to catastrophic telomere loss and stem cell exhaustion. EMBO J 31:2309-21|
|Sampathi, Shilpa; Chai, Weihang (2011) Telomere replication: poised but puzzling. J Cell Mol Med 15:3-13|
|Sampathi, Shilpa; Chai, Weihang (2011) Mapping the FEN1 interaction domain with hTERT. Biochem Biophys Res Commun 407:34-8|
|Dai, Xueyu; Huang, Chenhui; Bhusari, Amruta et al. (2010) Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection. EMBO J 29:2788-801|
|Sampathi, Shilpa; Bhusari, Amruta; Shen, Binghui et al. (2009) Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance. J Biol Chem 284:3682-90|