Project Summary/Abstract
This project is focused on the biology of telomeres, the protective repetitive sequences at chromosome ends
whose erosion determines the replicative lifespan of primary human cells. Telomere dysfunction has been
implicated in a number of human diseases, including inherited bone marrow failure syndromes and cancer. Our
aim is to understand how telomeres protect chromosome ends and how they are maintained. We have gained
insight into these issues through studies of shelterin, the six-subunit protein complex that specifically binds to
telomeric DNA. Shelterin is anchored on telomeres by two double-stranded telomeric DNA binding proteins,
TRF1 and TRF2, which promote telomere replication and protect telomeres from the DNA damage response,
respectively. This proposal focuses on these two critical telomere factors. In AIM 1. we will investigate the
function of TRF2. TRF2 is required for the formation of t-loops, the altered telomere structure formed by strand-
invasion of the 3’ telomeric overhang into the double-stranded telomeric DNA. The t-loop structure has been
proposed to prevent activation of the ATM kinase by hiding the telomere terminus from the Double-strand Break
(DSB) sensor (MRN) in the ATM pathway. Similarly, t-loops have been proposed to block the loading of Ku70/80
to prevent the c-NHEJ pathway from acting on chromosome ends. T-loop formation is thought to require the
TRFH domain of TRF2 but its mode of action is unclear. We are analyzing the biochemical aspects of the TRFH
domain and found novel features with likely relevance to t-loop formation that will be tested in vivo. In AIM 2, we
will test the role of t-loops in telomere protection. We are developing orthogonal tools for TRF2-independent t-
loop formation that will allow us to determine whether t-loops are sufficient to protect telomeres. In AIM 3, we will
investigate the function of TRF1. Loss of TRF1 induces telomere replication problems manifesting as fragile
telomeres, replication fork stalling, and sister telomere associations. Our data show how TRF1 uses the BLM
helicase to prevent lagging-strand replication problems caused by G4 DNA. We propose to investigate how
TRF1 prevents leading-strand replication problems, fork stalling, and sister telomere associations. We will
expand on our unpublished data indicating that the sister telomere associations represent a novel type of
telomere fusion resulting from a previously unrecognized alt-NHEJ mechanism involving stalled replication forks.
In AIM 4, we will study the function of the Myb domains of TRF1 and TRF2. Our unpublished data challenge the
widely-held view that Myb domains only function as DNA binding modules. We will pursue our hypothesis that
the Myb domains of TRF1 and TRF2 mediate unanticipated protein-protein interactions with a critical roles in
telomere protection and replication. The proposed experiments will use the biochemical, cell biological, genetic,
and molecular tools for telomere analysis developed in our lab in combination with innovative experimental
approaches. These experiments have the potential to lead to new concepts in telomere biology and are relevant
to the role of telomeres in age-related human disease states.