Structural insights can shape new thinking and contribute paradigm-shifting impact. Telomeres are central to cancer and aging. Questions of structure are central to telomere function where signaling to the DNA repair pathway likely depends on physical changes in the telomere. Previous work from this laboratory led to the discovery of telomere looping (t-loops) and small telomeric DNA circles (t-circles) which have now been found from yeast to humans and likely contributes to telomere maintenance. A new player discovered by others is telomeric RNA bound at the telomere. hnRNPA1 protein binds this RNA tightly and recent work in this laboratory revealed that hnRNPA1 will unwind duplex mammalian telomeric DNA. This research program applies a unique combination of biochemistry and transmission electron microscopy (EM). New EM tagging methods that identify proteins in multiprotein complexes have been developed and will be applied along with new ultra-gentle preparative methods, cryoEM and single particle reconstruction methods, melded with biochemical assays.
In AIM I, the core telomere binding proteins (TRF1, TRF2, Pot1, TPP1, hRap1, and Tin2) highly purified and in hand together with co-complexes of these proteins assembled in vivo will be assembled onto model telomere templates, their structure examined, and their ability to remodel telomeric DNA determined.
In AIM II, experiments using transgenic mice will further probe role of TRF2, and work on the Rad51 paralogs and the WRN helicase will be conducted to examine the interaction of these repair factors with the telomere complexes on telomeric DNA.
AIM III will focus on telomeric RNA and hnRNPA1 in their ability to form a scaffold at the telomere upon which other core telomere binding factors may assemble. The role of hnRNPA1 in facilitating looping and replicative extension of the telomere will be investigated.
Aim I V continues a long standing collaboration with the Tomaska group and the discovery of a novel new yeast species with long telomeres and telomere binding protein highly homologous to human TRF1/2. This yeast should provide a better model for human telomere biology. The high productivity of the past funding period provides a strong metric for future success and this is bolstered by strong collaborations. These studies have very high impact since no other laboratory is applying this technology to telomere/repair work and many other laboratories depend on the input from these structural studies. .

Public Health Relevance

Telomeres provide the first line of defense against cancer, and also provide a molecular clock that is central to ageing in human cells. Many basic questions of telomere structure and how these molecular machines signal to the DNA repair pathways remain unknown. The goal of this program is to provide answers to central questions that currently limit progress and thinking in this field.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01ES013773-09
Application #
8641689
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Heacock, Michelle
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sepsiova, Regina; Necasova, Ivona; Willcox, Smaranda et al. (2016) Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins. PLoS One 11:e0154225
Simonicova, Lucia; Dudekova, Henrieta; Ferenc, Jaroslav et al. (2015) Saccharomyces cerevisiae as a model for the study of extranuclear functions of mammalian telomerase. Curr Genet 61:517-27
Ciesielski, Grzegorz L; Bermek, Oya; Rosado-Ruiz, Fernando A et al. (2015) Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA. J Biol Chem 290:28697-707
Lewis, Samantha C; Joers, Priit; Willcox, Smaranda et al. (2015) A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans. PLoS Genet 11:e1004985
Dillon, Laura W; Kumar, Pankaj; Shibata, Yoshiyuki et al. (2015) Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and Transcriptional Activity. Cell Rep 11:1749-59
Ozgur, Sezgin; Griffith, Jack (2014) Interaction of Kaposi's sarcoma-associated herpesvirus ORF6 protein with single-stranded DNA. J Virol 88:8687-95
Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki et al. (2014) The strictly aerobic yeast Yarrowia lipolytica tolerates loss of a mitochondrial DNA-packaging protein. Eukaryot Cell 13:1143-57
Bower, Brian D; Griffith, Jack D (2014) TRF1 and TRF2 differentially modulate Rad51-mediated telomeric and nontelomeric displacement loop formation in vitro. Biochemistry 53:5485-95
Chen, Stefanie Hartman; Plank, Jody L; Willcox, Smaranda et al. (2014) Top3α is required during the convergent migration step of double Holliday junction dissolution. PLoS One 9:e83582
Lee, Shun-Hsiao; Siaw, Grace Ee-Lu; Willcox, Smaranda et al. (2013) Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases. Proc Natl Acad Sci U S A 110:E3587-94

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