TRF1 averts chromatin remodelling, recombination and replication dependent-break induced replication at mouse telomeres

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.

Characterization of t-loop formation by TRF2

“…The extensive proteomics efforts on TRF2 complexes and telomeric chromatin have not nominated obvious additional candidates (e.g. [ 36 – 40 ]. Therefore, the mystery of TRF2-mediated t-loop formation remains unsolved and other models should be pursued.…”

The human SKI complex prevents DNA-RNA hybrid-associated telomere instability

“…Cells were arrested in G1 with a double thymidine block, released and collected at different time points (Figures S1A and S1B). Telomere binding was examined using Proteomic of Isolated Chromatin segments (PICh) (Dejardin and Kingston, 2009;Porreca et al, 2020). The PICh analysis was conducted using unique peptides identified in each condition and the respective Label-free quantification (LFQ) values.…”

“…PICh experiments were conducted as previously described (Dejardin and Kingston, 2009;Porreca et al, 2020) with minor modifications on the sample processing:…”

“…pLKO.1 GAPDH shRNA construct (Open biosystems, 5'-CTCATTTCCTGGTATGACA-3')(Porreca et al, 2020), respectively. HeLa1.3 cells were transduced following standard procedures.…”

BLM-dependent Break-Induced Replication handles DSBs in transcribed chromatin upon impaired RNA:DNA hybrids dissolution

“…Indeed we found that (i) the depletion of POLD3 rescues lethality upon DSB in active genes in a SETX-depleted background, (ii) excessive DNA synthesis takes place at DSBs upon SETX depletion despite reduced RAD51 loading (Cohen et al, 2018), (iii) this repair-associated DNA synthesis depends on POLD3 and BLM, and (iv) BIR genomic signature correlates with expression levels of SETX, POLD3 and BLM in cancer patient samples. So far, POLD3-dependent BIR in human cells has been mainly involved in Alternative Lengthening of Telomeres (ALT) (Dilley et al, 2016;Min et al, 2019;Porreca et al, 2020;Roumelioti et al, 2016;Sobinoff et al, 2017;Zhang et al, 2019) and at one-ended DSBs, collapsed replication forks by a process also known as MiDAS (Mitotic Induced DNA synthesis) (Bhowmick et al, 2016;Costantino et al, 2014;Minocherhomji et al, 2015;Sotiriou et al, 2016). Moreover, BIR has been previously proposed to account for specifics genomic signatures, including tandem duplication, on the human genomes (Carvalho et al, 2013;Costantino et al, 2014;Hastings et al, 2009aHastings et al, , 2009bWillis et al, 2015;Zhang et al, 2009), suggesting that BIR indeed occurs in some context in mammalian cells.…”