FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres

Abstract

In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.aithfully replicating its genome is vital for the fitness and health of a mammalian cell. The replisome frequently encounters a variety of impediments throughout the genome. The temporary/transient slowing or stalling of the replication fork is referred to as replication stress (1, 2). The list of endogenous sources of replication stress is still growing. Nonetheless, the wellrecognized sources of replication stress include unrepaired DNA lesions, mis-incorporated ribonucleotides, unique DNA sequences that are prone to form secondary structures (e.g., G-quadruplex or G4), collision of the replication fork with the transcriptional machinery, an RNA-DNA hybrid (or R-loop) that is formed between a nascent RNA and the adjacent displaced single-stranded DNA (ssDNA), common fragile sites (CFS), and tightly packed genomic regions, such as heterochromatin (2). Because of these constant challenges faced by the replisome, mammalian cells have developed elaborate and complex strategies to resolve the replication stress and ensure the successful completion of DNA replication (1-4).One of the endogenous loci/regions that frequently pose challenges to the replisome is the telomere. Mammalian telomeres are tandem repetitive DNA sequences [the large majority as (TTAGGG) n ] located at the end of every linear chromosome. The addition of TTAGGG is catalyzed by an enzyme called telomerase, a large ribonucleoprotein complex with reverse tr...

Telomere length heterogeneity in ALT cells is maintained by PML-dependent localization of the BTR complex to telomeres

“…Depletion by siRNA in ALT-positive cells results in the reduction of ALT-associated phenotypes such as the accumulation of extrachromosomal telomeric repeats in the form of partially single-stranded Crich circles, termed C-circles, and G2/M telomere synthesis (O'Sullivan et al 2014;Pan et al 2019;Zhang et al). Notably, BLM also plays an important role at telomeres in cells that do not utilize ALT to maintain their telomeres, acting to facilitate telomere replication and suppressing rapid telomere deletions (Stavropoulos et al 2002;Sfeir et al 2009;Barefield and Karlseder 2012;Zimmermann et al 2014 ;Drosopoulos et al 2015;Pan et al 2017). BLM is part of the BTR complex which also includes the topoisomerase TOP3a, and the OB-fold containing structural components RMI1 and RMI2 (Johnson et al 2000;Wu et al 2000;Yin et al 2005;Xu et al 2008).…”

FANCM-family branchpoint translocases remove co-transcriptional R-loops

“…FANCM 119 could also remove R-loops formed by transcription through the telomeric repeat sequence, 120 otherwise known as TERRA transcripts(Figure 4b). These R-loops have previously been shown 121to have a strong G-quadraplex forming ability(Martadinata & Phan, 2013), suggesting that 122 FANCM's R-loop processing capability extends to R-loops that contain G-quadraplexes either 123 within the displaced strand or between the RNA and the displaced strand(Arora et al, 2014), 124 and supports a role for FANCM in the TERRA-dependent maintenance of telomeres by the ALT 125 pathway of telomere maintenance, Pan et al, 2017. 126As R-loops of greater than 600bp can be detected in a cellular context(Ginno et al, 2012) we 127 examined whether FANCM-FAAP24 could processively unwind R-loops containing different 128 lengths of RNA.…”

“…TERRA transcripts are produced by transcription of the C-rich telomeric DNA strand 259 and are essential for the ALT mechanism of telomere maintenance by recombination. FANCM is 260 necessary for ALT (Pan et al, 2017), and it is possible that this is because it acts on TERRA 261 transcripts to permit telomere replication or somehow regulate telomere recombination. Our in 262 vitro experiments demonstrate that FANCM displaces the G-quadraplex stabilized TERRA 263 transcripts as efficiently as it acts on promoter-or switch-region-based R-loops.…”

“…This was not because of increased transcription, but because of failure to properly remove R-30 loops (Schwab et al, 2015). Similarly, R-loops accumulate at telomeres after FANCM depletion, 31 particularly in cells that utilize the ALT pathway of telomere maintenance (Pan et al, 2017). As 32 such, FANCM may also prevent accumulation of DNA:RNA hybrids by the TERRA long non-33 coding RNA, which is essential for maintenance of ALT (Arora et al, 2014).…”

Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

“…Telomeres present many challenges to replisome progression including the propensity to form G-quadruplexes, R-and telomere-loops (t-loop) (Maestroni et al, 2017, Mason-Osann et al, 2019. Telomere replication not only requires canonical replisome proteins, but is facilitated by components of the Shelterin complex, DNA helicases, homologous recombination and DNA repair factors (Martinez and Blasco, 2015, Pan et al, 2017, Schmutz et al, 2017, Vannier et al, 2012. In contrast, the alternative lengthening of telomeres (ALT) pathway relies heavily on break-induced replication and has defined features that are distinct from telomere maintenance in telomerase-positive cells (Dilley et al, 2016, Sobinoff et al, 2017.…”