Telomeres pose a challenge to the replication machinery due to the repetitive nature of the DNA and its unusual terminal structure. As a result, telomere replication requires the action of ancillary factors in addition to the conventional replication machinery. Mechanistic understanding of how the actions of these factors are coordinated is still lacking and additional factors are being discovered. During the previous grant period the applicant identified a new telomere replication factor, the human CST (CTC1-STN1-TEN1) complex. CST has independent functions during telomere duplex replication and during fill-in synthesis of the telomeric C- strand after G-strand elongation by telomerase. CST also facilitates new origin firing during genome-wide replication restart after replication fork stalling. Moreover, mutations in the CTC1 subunit of CST cause dyskeratosis congenita and the neurological disorder Coats plus. CTC1 and STN1 correspond to AAF, a previously identified polymerase ? (pol ?) stimulatory factor. The objective of this proposal is to determine the mechanism of CST action during telomere replication and hence to understand how deficiencies in CTC1 lead to human disease. The central hypotheses is that CST enables pol ? to (re-)initiate DNA synthesis after replication fork stalling in the telomere duplex and during maturation of the telomeric 3'overhang. We propose that CST functions in conjunction with shelterin/TPP1 and telomeric DNA to recruit or load pol ? for C-strand fill-in at the 3'overhang and that CST facilitates new origin firing or fork restart within the telomere duplex.
Aim 1 will define CST structure and function by analysis of disease-related and DNA-binding mutations. Experiments will examine the effects of these mutations on telomere replication and CST biochemistry.
Aim 2 will determine how CST directs pol ? activity at the telomeric 3'overhang. In vitro studies will examine how CST activity is modified by telomere components while in vivo studies will examine the importance of CST, pol ? and TPP1 interactions for pol ?-mediated C-strand synthesis.
Aim 3 will determine how CST functions in telomere duplex replication. The replication barriers overcome by CST action will be defined and the pathway for replication rescue will be identified. Outcome: Collectively the three aims will provide essential insight into the mechanism of action of a novel telomere replication factor and will answer fundamental questions about how this factor prevents telomere loss and human disease. The work will reveal how CTC1 architecture impacts CST function and will uncover why CTC1 patient mutations cause Coats plus or dyskeratosis congenita. It will lead to a new understanding of how CST enables the cell to overcome replication barriers within the telomere to prevent telomere loss and how CST permits pol ? to complete replication of the chromosome terminus. The significance is that the work will provide key information about aspects of telomere replication that directly impact human health. This information is needed for better diagnosis and treatment of telomere-related disease.
The proposed research is relevant to public health because deficiencies in proteins necessary for telomere replication underlie a variety of human diseases including telomere syndromes such as dyskeratosis congenita. This project addresses the function of a newly identified telomere replication factor, CST, in which mutations have recently been shown to cause dyskeratosis congenita and the neurological disorder Coats plus. This work examines how CST functions during telomere replication and why CTC1 mutations cause disease.
|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|
|Price, Carolyn M (2012) Telomere flip-flop: an unfolding passage to senescence. EMBO Rep 13:5-6|
|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|
|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|
|Churikov, Dmitri; Price, Carolyn M (2008) Pot1 and cell cycle progression cooperate in telomere length regulation. Nat Struct Mol Biol 15:79-84|
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