FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres Xiaolei Pana, William C. Drosopoulosb, Louisa Sethia, Advaitha Madireddyb, Carl L. Schildkrautb, and Dong Zhanga,1 aDepartment of Biomedical Science, College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568; and bDepartment of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461 Edited by James E. Haber, Brandeis University, Waltham, MA, and approved June 13, 2017 (received for review May 16, 2017) In the mammalian genome, certain genomic loci/regions pose greater cells have to overcome the telomere dysfunction-induced cell cycle challenges to the DNA replication machinery (i.e., the replisome) than arrest or cell death by adopting one of the two telomere mainte- others. Such known genomic loci/regions include centromeres, com- nance mechanism (TMMs): (i)85–90% of cancers reactivate the mon fragile sites, subtelomeres, and telomeres. However, the detailed expression of telomerase (8), and (ii) the remaining 10–15% of mechanism of how mammalian cells cope with the replication stress at cancers use the HR-based ALT pathway (9, 10). these loci/regions is largely unknown. Here we show that depletion of The unique sequence and structure of mammalian telomeres FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, render them especially challenging to the replisome. First, one of and MHF2, induces replication stress primarily at the telomeres of cells the strands of telomere is rich in guanines (thus called the G-rich that use the alternative lengthening of telomeres (ALT) pathway as strand) and is prone to the formation of G4s (11, 12). Second, the their telomere maintenance mechanism. Using the telomere-specific subtelomeric/telomeric noncoding RNA, TERRA, was shown to single-molecule analysis of replicated DNA technique, we found that form an R-loop (13, 14). Third, we showed previously that DNA depletion of FANCM dramatically reduces the replication efficiency at replication can be initiated from the telomeres; however, the ALT telomeres. We further show that FANCM, BRCA1, and BLM are majority of the DNA replication near the end of a mammalian actively recruited to the ALT telomeres that are experiencing replication chromosome is initiated from the subtelomeres (15), suggesting stress and that the recruitment of BRCA1 and BLM to these damaged that the subtelomeric/telomeric regions lack DNA replication or- telomeres is interdependent and is regulated by both ATR and Chk1. igins. In addition, de Lange and colleagues observed that low Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, dosage of aphidicolin, a DNA polymerase inhibitor, dramatically BRCA1 and BLM help to resolve the telomeric replication stress by increases the incidence of fragile telomeres (16). Together, these stimulating DNA end resection and homologous recombination (HR). two features indicate that telomeres may be a special type of CFS. Consistent with their roles in resolving the replication stress induced by It is also known that ALT cells have more heterogeneous telo- FANCM deficiency, simultaneous depletion of BLM and FANCM, or of meres and that some of them can be quite long (17). Therefore, BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal ALT telomeres may be even more prone to express the fragility. interactions can be explored for targeting the ALT cancers. Fourth, heterochromatins are enriched at the telomeric and sub- telomeric regions of the genome (18). Finally, to protect the end – FANCM | BRCA1 | BLM | replication stress | ALT telomeres of linear chromosomes, there are a variety of high-order DNA DNA and DNA–protein structures at the telomeres. For example, the telomeric G-strand overhang folds back, invades the internal aithfully replicating its genome is vital for the fitness and double-stranded regions of telomere, and forms the telomeric health of a mammalian cell. The replisome frequently en- F loop (T-loop) as well as the displacement-loop (D-loop) (19, 20). counters a variety of impediments throughout the genome. The The T-loop and D-loop are further stabilized by the Shelterin temporary/transient slowing or stalling of the replication fork is complex and other proteins (21, 22). These various high-order referred to as replication stress (1, 2). The list of endogenous sources of replication stress is still growing. Nonetheless, the well- structures may also slow down the progression of the replisome. recognized sources of replication stress include unrepaired DNA lesions, mis-incorporated ribonucleotides, unique DNA sequences Significance that are prone to form secondary structures (e.g., G-quadruplex or G4), collision of the replication fork with the transcriptional ma- Currently, chemotherapy is still the only treatment option for chinery, an RNA–DNA hybrid (or R-loop) that is formed between ALT cancers. ALT cells rely heavily on homologous recombination a nascent RNA and the adjacent displaced single-stranded DNA to maintain their telomere length and are more susceptible to (ssDNA), common fragile sites (CFS), and tightly packed genomic replication stress. A better understanding of how ALT cells re- regions, such as heterochromatin (2). Because of these constant spond to replication stress at their telomeres will help to uncover challenges faced by the replisome, mammalian cells have developed novel therapeutic strategies to treat ALT cancers. Here we found elaborate and complex strategies to resolve the replication stress that depletion of FANCM or its obligatory binding partners in- and ensure the successful completion of DNA replication (1–4). duces pronounced replication stress at ALT telomeres. We thus One of the endogenous loci/regions that frequently pose chal- propose that FANCM-depleted ALT cells can be used as an en- lenges to the replisome is the telomere. Mammalian telomeres dogenous replication stress model. Most importantly, we pro- are tandem repetitive DNA sequences [the large majority as pose that the new synthetic lethal interactions discovered in our study can be explored for targeting ALT cancers. (TTAGGG)n] located at the end of every linear chromosome. The addition of TTAGGG is catalyzed by an enzyme called telomerase, Author contributions: X.P., W.C.D., L.S., A.M., C.L.S., and D.Z. designed research; X.P., a large ribonucleoprotein complex with reverse transcriptase activ- W.C.D., L.S., A.M., and D.Z. performed research; X.P., W.C.D., L.S., A.M., C.L.S., and D.Z. ity. The length of mammalian telomeres varies from 10 to 15 kb analyzed data; and X.P., C.L.S., and D.Z. wrote the paper. in humans and 25–50 kb in mice (5). Because most somatic cells The authors declare no conflict of interest. do not express telomerase, they experience progressive telomere This article is a PNAS Direct Submission. shortening with each cell cycle, which eventually leads to cellular 1To whom correspondence should be addressed. Email: [email protected]. senescence and aging of the organism (6). One of the hallmarks of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cancers is their replicative immortality (7). To achieve that, cancer 1073/pnas.1708065114/-/DCSupplemental. E5940–E5949 | PNAS | Published online July 3, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1708065114 Downloaded by guest on September 27, 2021 Taken together, mammalian telomeres pose constant challenges single-molecule analysis of replicated DNA (SMARD) tech- PNAS PLUS to the replisome, and replication stress often takes place at the nique, we found that depletion of FANCM dramatically reduced telomeres, especially in ALT cells (16, 23, 24). Therefore, a robust the replication efficiency at ALT telomeres. We therefore propose replication stress response is vital for the successful replication that depletion of FANCM in ALT cells can be used as an en- of telomeres. dogenous replication stress model. Mechanistically, we found that, FANCM is an evolutionarily conserved ATP-dependent DNA in this endogenous replication stress model, BLM and BRCA1, helicase/translocase (25, 26). It belongs to the Fanconi anemia two of the most important HR proteins, are actively recruited to (FA) family of genes, the most recognized function of which is to the replication stress sites and help to resolve the replication stress repair the interstrand cross-linking (ICL) lesions (27, 28). FANCM by stimulating DNA end resection and promoting HR. Finally, we and its three obligatory binding partners, FAAP24, MHF1, and found that codepletion of FANCM and BLM or of FANCM and MHF2, are part of the FA core complex, which catalyzes the BRCA1 dramatically increases the micronuclei formation and monoubiquitination of FANCD2 and FANCI (ID2) (29–33). In induces synthetic lethality in ALT cells. addition, FANCM can also promote checkpoint activation and replication traversal across the ICL lesion (34–37). Furthermore, Results FANCM also plays a critical role in replication stress response. For Depletion of FANCM Induces Replication Stress at ALT Telomeres. example, in cells treated with hydroxyurea (HU), FANCM binds Based on the potential roles of FANCM in DNA replication, the checkpoint protein HCLK2 and promotes the activation of we hypothesized that the DNA damages seen in FANCM- ATR-mediated checkpoint response (34). FANCM also helps to deficient cells are due to replication stress (34, 38). Replication stabilize