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. .
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.
|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|
|Tolun, Gokhan; Makhov, Alexander M; Ludtke, Steven J et al. (2013) Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex. Nucleic Acids Res 41:5927-37|
|Amunugama, Ravindra; He, Yujiong; Willcox, Smaranda et al. (2012) RAD51 protein ATP cap regulates nucleoprotein filament stability. J Biol Chem 287:8724-36|
|Shibata, Yoshiyuki; Kumar, Pankaj; Layer, Ryan et al. (2012) Extrachromosomal microDNAs and chromosomal microdeletions in normal tissues. Science 336:82-6|
|Wu, Congying; Asokan, Sreeja B; Berginski, Matthew E et al. (2012) Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis. Cell 148:973-87|
|Kramara, Juraj; Willcox, Smaranda; Gunisova, Stanislava et al. (2010) Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica. J Biol Chem 285:38078-92|
|Compton, Sarah A; Ozgur, Sezgin; Griffith, Jack D (2010) Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy. J Biol Chem 285:13349-56|
|Basenko, Evelina Y; Cesare, Anthony J; Iyer, Shilpa et al. (2010) Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination. Nucleic Acids Res 38:182-9|
|Randall, Adrian; Griffith, Jack D (2009) Structure of long telomeric RNA transcripts: the G-rich RNA forms a compact repeating structure containing G-quartets. J Biol Chem 284:13980-6|
|Remus, Dirk; Beuron, Fabienne; Tolun, Gokhan et al. (2009) Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139:719-30|
Showing the most recent 10 out of 22 publications