Mechanistic Insights into the Structural Basis for End Replication in Humans and End Protection in C. elegans
Padmanaban, Shilpa
2023
Abstract
Telomeres are nucleoprotein complexes at natural ends of chromosomes crucial for preserving genomic stability in eukaryotes. The ends of chromosomes can be misrecognized as DNA double-stranded breaks by the DNA damage response machinery. Deprotection of chromosome ends could result in deleterious end-to-end fusions. A protein complex called shelterin binds to the telomeric DNA to protect them from illicit DNA repair. Telomeres also undergo shortening with each cell division because DNA polymerases cannot synthesize the very ends of chromosomes. The reverse transcriptase, telomerase, maintains telomere ends and is recruited by shelterin protein TPP1. Telomerase is only expressed in stem cells and in ~90% of cancers to replicate chromosome ends and enable continued cell division. In 4-11% of cancer cells, alternative lengthening of telomeres (ALT) is used to maintain telomeres. The nematode, C. elegans, is a unique model system to study ALT in a non-disease context. Here, I present new insights into telomere maintenance using human telomerase and C. elegans shelterin. I dissect the structural features of human telomerase that differentiates it from other reverse transcriptases and ensures action only at telomere ends. I also use C. elegans as a model system to investigate the structural features that allow telomere end-binding proteins (TEBPs) TEBP-1 and TEBP-2 to protect C. elegans telomeric DNA. Domain deletions and mutational analysis of human telomerase probed using enzymology and human cell biology reveal critical interactions between the catalytic protein subunit TERT, the RNA subunit TR, the DNA primer, and the shelterin component TPP1. TERT-specific domains, TEN and IFD-TRAP, engage in a three-way interaction with TPP1, facilitating efficient telomerase recruitment and processive telomere repeat addition. Disrupting the IFD-TRAP’s interface with TR or the DNA primer through single mutations impairs telomerase catalysis. Moreover, TERT elements, including motif 3N and the TEN domain loop 99FGF101, play crucial roles in orchestrating telomerase action. Together, this study identifies novel structural features that are critical for human telomerase recruitment and catalysis. Using C. elegans as a model system to study end protection, I dissect the dual functionalities of recently identified shelterin proteins TEBP-1 and TEBP-2 in DNA binding and dimerization, and how they contribute to protecting telomeres. X-ray crystallography and biochemistry reveal that TEBP-1 and TEBP-2 employ Myb-containing domains (MCDs) for binding telomeric DNA, and surprisingly, for dimerization. I provide structural insights into how MCD2 is used to bind DNA and highlight conserved and distinct features when compared to their mammalian counterparts, TRF1/2, in their interactions with DNA. While Myb domains are best known as DNA-binding domains, MCD1 and MCD2 domains of TEBP-1 and TEBP-2 mediate both homodimerization and heterodimerization, unlike TRF1/2 that use a “TRFH” domain to just homodimerize. This study advances C. elegans telomere biology and highlights how different eukaryotes use distinct structural mechanisms to reach the same end goal of chromosome end protection. Collectively, my Ph.D. dissertation work expands our knowledge of telomere biology in different eukaryotic models by illuminating molecular mechanisms by which they protect and replicate their chromosome ends.Deep Blue DOI
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Telomeres Telomerase
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