Combining conservation and species-specific differences to determine how human telomerase binds telomeres

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Telomerase is crucial for maintaining telomere length and enabling continuous cell division.
• The telomeric protein TPP1 enhances telomerase processivity and recruits it to telomeres.
• Previous studies mapped the TPP1 interaction surface to its N-terminal oligosaccharide/oligonucleotide-binding (OB) domain.

Purpose of the Study:

• To map the telomerase-interacting regions of TPP1.
• To identify specific amino acid residues on telomerase reverse transcriptase (TERT) critical for TPP1 binding.
• To develop a structural model of the human telomerase-TPP1 interface.

Main Methods:

• Exploited structural similarities between human and Tetrahymena thermophila telomerase.
• Utilized species-specific interactions between human and mouse telomerase and TPP1.
• Performed domain swapping experiments with human and mouse TERT.
• Conducted homology-based mutagenesis screens on TERT surface residues.

Main Results:

• Swapping the N-terminal extension (TEN) and insertions in fingers domain (IFD)-TRAP regions of human TERT into mouse TERT biased species specificity towards human TPP1.
• Identified critical TERT residues in the TEN and IFD regions essential for TPP1 interaction.
• These identified residues are dispensable for other telomerase structural or functional aspects.

Conclusions:

• The TEN and IFD regions of TERT are key determinants of TPP1 binding specificity.
• A functional structural model for human telomerase engaging TPP1 at telomeres was established.
• This work lays the foundation for high-resolution structural studies of the telomerase-TPP1 interface.