Telomeric Repeats Induce Replication Fork Reversal In Vivo

Huda, Armela (2021). Telomeric Repeats Induce Replication Fork Reversal In Vivo. PhD thesis The Open University.



Telomeres are the terminal parts of linear chromosomes made of tandem repeats of the TTAGGG motif. Telomeric repeats are associated with a six-protein complex called shelterin, which protects chromosome ends from the DNA Damage Response and regulates their maintenance. Telomeres are hard to replicate and behave like replication fragile sites and telomere replication constitutively requires shelterin and specialized helicases involved in the replication stress response. The repetitive nature of the telomeric repeats and the tendency to form secondary structures like G4 DNA, are thought to challenge fork progression at telomeres, however the molecular bases of telomere replication problems are not fully understood, in part due to the lack of techniques that can monitor structural transitions at telomeric replication forks. To overcome this limitation, we introduced telomeric repeats with different lengths and orientations, in an SV40-based construct that has been previously used to study the replication of specific DNA sequences in mammalian cells. Consistent with previous studies, we show that the shelterin complex associates with the telomeric repeats in our constructs, mimicking the sequence context of natural telomeres. Introduction of an episomal construct does not interfere with the structure of natural telomeres. Two-dimensional agarose gel electrophoresis showed that as telomeric insertions increase in length, they accumulate X-shaped intermediates compared to non-telomeric DNA sequences, independently of the orientation of the telomeric repeats with respect to the SV40 replication origin. In order to define the exact molecular nature of the intermediates that accumulate at telomeric repeats, we isolated replication intermediates and analyzed them in Electron Microscopy. Consistent with the 2D-gel analysis, we found that fragments containing 115 telomeric repeats have a 2-fold increase in replication fork reversion compared to control DNA sequences. These results show that replication forks have a higher probability of undergoing reversion at telomeric repeats in vivo.

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