The impact of telomeric components on a reconstituted human replisome
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Embargo End Date
2025-07-06
ICR Authors
Authors
Leonard-Booker, C
Document Type
Thesis or Dissertation
Date
2025-01-06
Date Accepted
Abstract
Eukaryotes face two problems in having linear chromosomes: i) the end-protection problem, where linear ends could be recognised as double strand breaks, undergoing chromosome end-end fusions and ii) the end-replication problem, defined as the loss of genetic information each round of cell division. Cells have evolved a specialised nucleoprotein structure called a telomere to counteract these problems. In humans, telomeric DNA is composed of 5-15 kilobases of TTAGGG repeats with a G-rich 3’ overhang and is bound by the multi-subunit complex shelterin that suppresses DNA damage signalling and repair mechanisms at telomeres. The enzyme telomerase can be recruited to add telomeric repeats onto the 3’ end but the main way telomeres are duplicated each cell cycle is canonical DNA replication. A failure to replicate telomeres can drive genome instability due to the loss of telomeric repeats. Despite the importance of accurate telomere replication, they are a source of replication stress. Indirect genetic evidence mainly attributes this to G-rich telomeric DNA, but the pleiotropic effects observed upon genetic deletions and the presence of other telomeric components like shelterin, complicates interpretation of this data.
This study aims to reconstitute a human replisome from purified proteins to analyse the impact of individual telomeric components on the replication process. Through this, I uncover that G-rich and C-rich telomeric DNA has minimal impact on the replication process whereas, shelterin can terminally stall the replisome similarly to other protein roadblocks. I reveal that shelterin induces a single-stranded gap specifically on the lagging strand, which is dependent upon a subcomplex of shelterin, containing protection of telomeres 1 (POT1) and its binding partner TPP1. Collectively, I uncover a dual inhibitory effect of shelterin on the replisome: i) fork stalling, and ii) defective lagging strand synthesis, providing key mechanistic insights into how shelterin interfaces with the replisome at telomeres.
Citation
2025
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Telomere Biology