Abstract
Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. Among the many prominent features of ALT cells, C-circles (CC) formation is considered to be the most specific and quantifiable biomarker of ALT. However, the molecular mechanism behind the initiation and maintenance of CC formation in ALT cells is still largely unknown. We reported previously that depletion of the FANCM complex (FANCM-FAAP24-MHF1&2) in ALT cells induced pronounced replication stress, which primarily takes place at their telomeres. Here, we characterized the changes in ALT associated phenotypes in cells deficient of the FANCM complex. We found that depletion of FAAP24 or FANCM, but not MHF1&2, induces a dramatic increase of CC formation. Most importantly, we identified multiple DNA damage response (DDR) and DNA repair pathways that stimulate the dramatic increase of CC formation in FANCM deficient cells, including the dissolvase complex (BLM-TOP3A-RMI1/2, or BTR), DNA damage checkpoint kinases (ATR and Chk1), HR proteins (BRCA2, PALB2, and Rad51), as well as proteins involved in Break-Induced Replication (BIR) (POLD1 and POLD3). In addition, FANCD2, another Fanconi Anemia (FA) protein, is also required for CC formation, likely through promoting the recruitment of BLM to the replication stressed ALT telomeres. Finally, we demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. Taken together, our data suggest that FANCM prevents replisomes from stalling/collapsing at ALT telomeres by disrupting TERRA R-loops.
Highlights
Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway
We demonstrated that depletion of FANCM and three of its binding partners, FAAP24, MHF1, and MHF2, in ALT cells induced different severity of replication stress at their telomeres[39]
In addition to the dissolvase complex, we found that two important checkpoint kinases (ATR and Chk1), three HR proteins (BRCA2, PALB2, and Rad51), two proteins involved in Break-Induced Replication (BIR) (PolD3 and PolD1), a translesion DNA polymerases, Polη, as well as a key Fanconi Anemia protein, FANCD2, stimulate the CC formation in FANCM deficient U-2 OS
Summary
Cancer cells maintain their telomeres by either re-activating telomerase or adopting the homologous recombination (HR)-based Alternative Lengthening of Telomere (ALT) pathway. We demonstrated that TERRA R-loops accumulate at telomeres in FANCM deficient ALT cells and downregulation of which attenuates the ALT-associated PML bodies (APBs), replication stress and CC formation. The telomeric G-rich overhang folds back, invades the double-stranded region of telomeres, and forms the telomeric loop (T-loop) and the displacement loop (D-loop)[7,8], which are facilitated and stabilized by a multi-subunit protein complex called Shelterin[9,10] All of these DNA-DNA, DNA-RNA, and DNA-protein structures need to be overcome in order for replisomes to successfully replicate telomeres during each cell cycle. Over-expression of the S. cerevisiae MPH1 gene, the yeast homolog of human FANCM, strongly suppresses the BIR at certain double-stranded breaks (DSBs)[25]
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