Telomeres provide the protective cap to the ends of linear
chromosomes. The regulation of telomere length is essential in
maintaining genome stability. Impairing the telomere
maintenance leads to dysfunctional telomeres, resulting in
telomere shortening associated with diseases such as bone
marrow failure, premature aging, and cancer. Telomere
maintenance is dependent on strict coordination between
telomeric protein complexes, telomerase, and telomeric DNA.
Shelterin represents the major protein complex that binds
telomeric DNA with a high affinity and sequence specificity.
Moreover, shelterin directly recruits numerous
telomere-associated factors, including telomerase, and is
responsible for shaping the telomeric structure and guarding
telomeres against DNA damage response machinery. The assembly
and cooperation of human shelterin proteins can regulate the
functions of the whole complex. Therefore, the complex
formation through dynamic interactions between shelterin
subunits is essential in telomere maintenance.
To elucidate the molecular mechanism of human shelterin
assembly, we focused on the telomeric repeat binding factors
TRF1 and TRF2, which together with TIN2 (TERF1-interacting
nuclear factor 2) form the core of the shelterin complex. We
applied a single-molecule approach based on fluorescence
cross-correlation spectroscopy (FCCS) to monitor the
spontaneous intensity fluctuations as the differently
fluorescently labeled TRF1/TRF2 and TIN2 diffuse through the
confocal volume of a microscope. The cross-correlation analysis
of measured intensity traces enabled us to understand the
TRF1-TIN2-TRF2 complex formation. We found that TRF1 can
effectively replace TRF2 bound to TIN2. In contrast, TRF2 had
no evident effect on the TRF1-TIN2 complex. Furthermore, we
assessed the effect of shelterin factor TPP1 (adrenocortical
dysplasia protein homolog) in promoting whole shelterin
complexation. Strikingly, TPP1 addition expanded the TIN2
binding capacity and allowed TIN2 to accommodate both TRF1 and
TRF2 simultaneously. We proposed a model describing the
assembly of human shelterin core subunits in solution. We
highlighted the TPP1 binding to TIN2 as an essential step in
stable shelterin formation. Moreover, we suggested the
TPP1-TIN2 interaction as the potential weakest point in
shelterin assembly, hence the critical step in telomere
maintenance.
The thesis outcomes contribute to understanding human shelterin
assembly and describe the role of TPP1 in promoting the whole
shelterin complexation. The obtained findings address the
cooperation of full-length telomeric proteins essential in
telomere maintenance at the single-molecule level. In addition,
the FCCS-based single-molecule platform served for
methodologically related studies, where we characterized the
practical impacts of deuterated water in fluorescence-based
methods.