Abstract
Telomeres are a significant challenge to DNA replication and are prone to replication stress and telomere fragility. The shelterin component TRF1 facilitates telomere replication but the molecular mechanism remains uncertain. By interrogating the proteomic composition of telomeres, we show that mouse telomeres lacking TRF1 undergo protein composition reorganisation associated with the recruitment of DNA damage response and chromatin remodellers. Surprisingly, mTRF1 suppresses the accumulation of promyelocytic leukemia (PML) protein, BRCA1 and the SMC5/6 complex at telomeres, which is associated with increased Homologous Recombination (HR) and TERRA transcription. We uncovered a previously unappreciated role for mTRF1 in the suppression of telomere recombination, dependent on SMC5 and also POLD3 dependent Break Induced Replication at telomeres. We propose that TRF1 facilitates S-phase telomeric DNA synthesis to prevent illegitimate mitotic DNA recombination and chromatin rearrangement.
Highlights
Telomeres are specialised nucleoprotein structures at the ends of chromosomes, composed of repetitive sequences (TTAGGG repeats in mammals) (Moyzis et al, 1988), long non-coding RNA called TERRA and six associated proteins, TRF1, TRF2, POT1a/b, RAP1 and TIN2, that form the shelterin complex (de Lange, 2005)
Based on the analysis of label free quantification (LFQ intensities), we found 119 proteins presenting a gain of abundance at TRF1 depleted telomeres (Log2-2), considering that a cut-off for differential expression is set to log2 fold change (TRF1deletion/wt)> |2| and -Log (p-value) > 1
Since the SMC5/6 complex was exclusively enriched in Proteomics of Isolated Chromatin segments (PICh) purified TRF1 depleted telomeres (Figure 2A), we further investigated the role of POLD3 and SMC5 in Break Induced Replication (BIR) DNA synthesis observed in TRF1-/- MEFs
Summary
Telomeres are specialised nucleoprotein structures at the ends of chromosomes, composed of repetitive sequences (TTAGGG repeats in mammals) (Moyzis et al, 1988), long non-coding RNA called TERRA and six associated proteins, TRF1, TRF2, POT1a/b, RAP1 and TIN2, that form the shelterin complex (de Lange, 2005). We found that telomeres lacking TRF1 are enriched in SMC5/6, DNA polymerase d (POLD3), and chromatin remodelling factors known to be associated with ALT telomeres These cells present additional DNA damage and recombination hallmarks such as formation of APBs, mitotic DNA synthesis at telomeres, a feature of BIR and increased TERRA levels. Further investigation using specific shRNAs against the SMC5/6 complex or POLD3 revealed how these two complexes are key regulators of the recombination signature identified in TRF1 deleted cells Taken together, these results strongly identify TRF1 as a central player in preserving telomeric chromatin against HR, induced by DNA replication stress, and POLD3 dependent-mitotic DNA synthesis
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