Elucidating the structure function relationship between TPP1 and telomere length control through deep scanning mutagenesis

University Of California Berkeley, Berkeley CA

Investigators

Abstract

ABSTRACT Telomeres are tandem TTAGGG nucleotide repeats that cap the linear ends of human chromosomes. They protect genetic information from gradual sequence loss due to incomplete chromosome end replication and processing by nucleases. The shelterin complex binds to telomeric DNA and regulates the elongation of telomeres by the enzyme telomerase. TPP1, a central component of this complex, recruits telomerase to telomere ends and is therefore required for telomere length maintenance. However, TPP1 also limits excessive telomerase activity by bringing the shelterin protein POT1 to telomeres. Mutations in TPP1 that result in telomere shortening cause the bone marrow failure syndrome dyskeratosis congenita, while mutations that elongate telomeres have recently been shown to cause cancer. The fact that mutations in the same protein can cause dramatically opposing effects on telomere length and disease poses a central challenge to the clinical evaluation of TPP1 mutations. There are currently over 650 variants of uncertain significance listed in CLINVAR whose effects on telomere length are unknown. The central goal of our experiments is to deploy deep scanning mutagenesis of TPP1 to comprehensively characterize TPP1 mutations and perform a detailed structure-function analysis to identify mutations that drive telomere length changes. Moreover, we seek to uncover the molecular mechanisms governing length-dependent extendibility by telomerase by identifying and characterizing TPP1 separation-of-function mutations that alter TPP1 abundance at telomeres without leading to telomere deprotection. Our experiments will test the hypothesis that TPP1 density defines telomerase-extendible or non-extendible telomeric states to establish the normal telomere length setpoint in stem cells and disrupting TPP1 density can cause human disease.