Summary
Telomeres are repetitive DNA and associated proteins at the ends of the chromosomes that are important for
genome stability. The shelterin complex, a six-member group of proteins, is important for telomere length
maintenance, which occurs through regulating the recruitment and action of the telomerase enzyme. In addition,
shelterin also protects the chromosome ends from being incorrectly recognized by DNA damage repair
mechanisms. Disorders in telomere biology are an underlying cause of many human diseases. Mutation of
telomerase or shelterin components, resulting in loss of telomere maintenance, leads to dyskeratosis congenita,
pulmonary fibrosis and aplastic anemia. Its medical importance is further shown by the fact that 90% of cancers
depend on hyper-activation of telomerase for persistent proliferation. Despite the critical roles shelterin plays in
genome stability, a consistent framework of understanding its mechanism in telomere maintenance has not been
established. A leading model of how shelterin can protect chromosome ends and restrict telomerase access to
the telomeric tail is the telomere-loop model. Other simpler models include DNA end-capping or compaction by
shelterin to form reclusive structures. While these models are derived from the DNA remodeling properties of
individual shelterin proteins, the collective roles shelterin proteins play as a functional multisubunit complex are
still ambiguous. How a shelterin complex organizes telomeric DNA into various architectures for their regulatory
functions and how they switch between inhibitory and permissive roles in telomerase elongation of telomeres
are the next key questions. The answers lie in the molecular mechanisms of these processes and hence, the
goal of this proposal is to elucidate the structural basis of shelterin complex functions in regulating telomerase
recruitment and elongation of telomeres. A multiscale and interdisciplinary approach will be used, which includes
biochemistry, biophysics, cell biology and cryo-EM techniques. Specifically, this proposal aims to determine: (1)
How the shelterin complex organizes telomeric DNA into various architectures that regulate telomerase
accessibility, (2) The structure of the shelterin complex assembled with telomeric DNA, and (3) How a shelterin
complex switches from telomere end-capping to telomerase stimulation. During the K99 phase, under the
mentorship of Dr. Tom Cech, AFM imaging with biochemical assays will be used to characterize higher-order
DNA-protein architectures formed by various shelterin complexes, and their effects on telomerase recruitment
and activity will be measured. With additional support from Dr. Zhiheng Yu, a cryo-EM skill set will be acquired
while determining the cryo-EM structure of shelterin in association with telomeric DNA. This will facilitate using
cryo-EM to study the structural basis of telomerase regulation by shelterin during the independent R00 phase.
The results of this proposal would provide a multiscale framework for understanding the mechanisms of shelterin
functions in telomere maintenance, and also potentially provide new avenues in developing therapeutic and
diagnostic strategies to combat human diseases of telomere dysfunction.
