The long term goal of this research is to explain how telomeres perform their various roles in cells. At present we know that telomeres are essential for proper cell growth and development. However, we only have limited information about telomere structure and we do not understand the relationship between their structure and their function. Hypotrichous ciliates (Euplotes and Oxytricha) will be used as the experimental system because these organisms have sufficient telomeric material to perform structural studies; their macronuclei contain literally millions of telomeres. Three complimentary approaches will be used to examine the importance of specific features of telomere structure during certain cellular events. Firstly, the structure of the telomeric complex from Euplotes crassus will be studied. The DNA-protein interactions will be analyzed by chemical and nuclease footprinting. The protein components of the complex will be identified and their DNA-binding properties studied. The structural features of the Euplotes and the Oxytricha nova telomeric complexes will be compared so that conserved features of possible functional importance can be identified. Secondly, de novo telomere formation will be studied during macronuclear development. As Euplotes crassus will mate synchronously, one can obtain large numbers of macronuclei at specific stages in development. By studying telomere structure during the various stages in development, it should be possible to identify the steps that lead to formation of the mature telomeric complex. These studies should clarify which structural features are required in certain cellular events, and should elucidate how the various components of a mature telomere interact with each other to form a functional structure. Lastly, changes in telomere structure associated with DNA replication will be studied. These studies should clarify how telomeres function during replication. Pre-replicating DNA molecules will be obtained from isolated replication bands. Newly- replicated molecules will be obtained by pulse labeling with biotinylated dUTP and affinity purifying the labeled molecules. Differences in DNA sequence and structure. DNA-protein interactions, and protein composition will be sought.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM041803-05
Application #
2181063
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1989-04-01
Project End
1994-11-30
Budget Start
1993-04-01
Budget End
1994-11-30
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Nebraska Lincoln
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
Wang, Feng; Stewart, Jason; Price, Carolyn M (2014) Human CST abundance determines recovery from diverse forms of DNA damage and replication stress. Cell Cycle 13:3488-98
Kasbek, Christopher; Wang, Feng; Price, Carolyn M (2013) Human TEN1 maintains telomere integrity and functions in genome-wide replication restart. J Biol Chem 288:30139-50
Wang, Feng; Stewart, Jason A; Kasbek, Christopher et al. (2012) Human CST has independent functions during telomere duplex replication and C-strand fill-in. Cell Rep 2:1096-103
Stewart, Jason A; Chaiken, Mary F; Wang, Feng et al. (2012) Maintaining the end: roles of telomere proteins in end-protection, telomere replication and length regulation. Mutat Res 730:12-9
Stewart, Jason A; Wang, Feng; Chaiken, Mary F et al. (2012) Human CST promotes telomere duplex replication and general replication restart after fork stalling. EMBO J 31:3537-49
Price, Carolyn M (2011) Telomere flip-flop: an unfolding passage to senescence. EMBO Rep 13:5-6
Baumann, Peter; Price, Carolyn (2010) Pot1 and telomere maintenance. FEBS Lett 584:3779-84
Price, Carolyn M; Boltz, Kara A; Chaiken, Mary F et al. (2010) Evolution of CST function in telomere maintenance. Cell Cycle 9:3157-65
Linger, Benjamin R; Price, Carolyn M (2009) Conservation of telomere protein complexes: shuffling through evolution. Crit Rev Biochem Mol Biol 44:434-46
Price, Carolyn M (2009) Antics at the telomere: uncoupled polymerases solve the end replication problem. EMBO J 28:795-6

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