The overall goal of this project is to understand how telomeres prevent the activation of the DNA damage response at chromosome ends. In the prior funding period we have made progress on defining this end- protection problem, establishing that the two main DNA damage response pathways, the ATM and ATR kinase signaling cascades, can be activated at chromosome ends when telomere protection is lost. In this proposal, we describe experiments designed to gain mechanistic insights into how telomeres repress the ATM and ATR kinases. Specifically, AIMs 1 and 2 will focus on two components of the telomeric shelterin complex, TRF2 and POT1, which we have shown to act independently to repress the ATM and ATR pathways at chromosome ends.
The third aim of this proposal is to examine how telomeres are duplicated by semi-conservative DNA replication. Our preliminary data indicates that telomeric repeats pose a considerable challenge to the replication fork, leading to replication fork arrest and phenotypes consistent with telomeres representing fragile sites. Thus, the data suggest that the protective telomeric DNA at chromosome ends comes at the cost of potential replication problems. TRF1, a third component of shelterin identified in our laboratory, was found to have a key role in facilitating the replication of telomeres. This new insight forms the basis of the work in AIM 3 where we will examine telomere replication in detail and address how TRF1 facilitates replication fork progression through the telomeric DNA. Together these experiments should reveal the basic principles by which the telomeric shelterin complex allows mammalian cells to maintain linear chromosomes without activating DNA damage signaling cascades. Human cells lose telomeric DNA with cell divisions and exhibit limited proliferation due to the eventual loss of telomere function. Understanding telomere function and the consequences of telomere deprotection is directly relevant to the limited life-span of primary human cells in vitro and will shed light on the finite ability of stem cells to replenish organ function in vivo. This research is therefore expected to improve the understanding of telomere- related diseases, such as dyskeratosis congenita, and diminished organ function with aging in normal individuals. Furthermore, the outcome of our experiments are relevant to human cancer since loss of telomere function is thought to be a driving force in the early stages of human tumorigenesis.

Public Health Relevance

We propose to define the mechanism by which telomeres protect the ends of human chromosomes. Telomere function is a key issue in human health because telomeres are lost with cell divisions and with age, resulting in diminished cell growth and impaired organ homeostasis. In several human diseases, the loss of telomeric DNA is exacerbated, leading to premature organ failure and death.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG016642-14
Application #
8293196
Study Section
Cellular Mechanisms in Aging and Development Study Section (CMAD)
Program Officer
Guo, Max
Project Start
1999-05-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
14
Fiscal Year
2012
Total Cost
$494,072
Indirect Cost
$201,721
Name
Rockefeller University
Department
Anatomy/Cell Biology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Frescas, David; de Lange, Titia (2014) A TIN2 dyskeratosis congenita mutation causes telomerase-independent telomere shortening in mice. Genes Dev 28:153-66
Frescas, David; de Lange, Titia (2014) TRF2-tethered TIN2 can mediate telomere protection by TPP1/POT1. Mol Cell Biol 34:1349-62
Zimmermann, Michal; de Lange, Titia (2014) 53BP1: pro choice in DNA repair. Trends Cell Biol 24:108-17
Kabir, Shaheen; Hockemeyer, Dirk; de Lange, Titia (2014) TALEN gene knockouts reveal no requirement for the conserved human shelterin protein Rap1 in telomere protection and length regulation. Cell Rep 9:1273-80
Frescas, David; de Lange, Titia (2014) Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection. J Biol Chem 289:24180-7
Doksani, Ylli; Wu, John Y; de Lange, Titia et al. (2013) Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell 155:345-56
Ahmed, Emad A; Sfeir, Agnel; Takai, Hiroyuki et al. (2013) Ku70 and non-homologous end joining protect testicular cells from DNA damage. J Cell Sci 126:3095-104
Takai, Kaori K; Kibe, Tatsuya; Donigian, Jill R et al. (2011) Telomere protection by TPP1/POT1 requires tethering to TIN2. Mol Cell 44:647-59
Scherthan, Harry; Sfeir, Agnel; de Lange, Titia (2011) Rap1-independent telomere attachment and bouquet formation in mammalian meiosis. Chromosoma 120:151-7
Davoli, Teresa; de Lange, Titia (2011) The causes and consequences of polyploidy in normal development and cancer. Annu Rev Cell Dev Biol 27:585-610

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