Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52

Abstract

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.

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

“…APBs have been proposed to play a critical role in ALT by clustering telomeres and DNA repair factors together, thus concentrating substrates and enzymes required for recombination-based telomere elongation (Henson et al 2002;Cesare and Reddel 2010;Chung et al 2011;Chung et al 2012;Min et al 2019;Verma et al 2019). In agreement with this hypothesis, it has recently been shown that telomeres can be elongated during mitosis in APB-like foci in a process termed mitotic DNA synthesis MiDAS (Ozer et al 2018;Min et al 2019). Moreover, depletion of PML by siRNA has been shown to reduce telomere elongation (Osterwald et al 2015).…”

“…Recent work in mammalian cells has paralleled the work done in yeast, revealing that ALT-positive cancer cells display a type I-like ALT mechanism that is RAD51-dependent and characterized by telomere clustering and recombination-mediated telomere synthesis (Cho et al 2014;Ramamoorthy and Smith 2015). In addition, ALT-positive mammalian cells also display RAD51-independent type II-like mechanisms of telomere elongation characterized by telomere synthesis in the G2/M phase of the cell cycle Min 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). Interestingly, overexpression of BLM or dysregulation of the BTR complex induced by the loss of FANCM in ALT-positive cells has been shown to induce upregulation of ALT-associated phenotypes, suggesting that this factor is limiting for the ALTpathway and led to the proposal that the BTR complex acts in ALT to dissolve recombination intermediates into non-crossover products which results in telomere lengthening (Sobinoff et al 2017;Lu et al 2019;Min et al 2019;Pan et al 2019;Silva et al 2019).…”

“…Cancer cells that maintain telomeres using the ALT pathway harbor unique features that are used as ALT biomarkers such as large promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA, termed ALT-associated PML-bodies (APBs), extrachromosomal telomeric DNA in the form of C-circles, elevated levels of telomere-sister chromatid exchanges (T-SCEs) and highly heterogenous telomere lengths (Ogino et al 1998;Tokutake et al 1998;Yeager et al 1999;Henson et al 2002;Cesare and Griffith 2004;Londono-Vallejo et al 2004;Wang et al 2004;Henson et al 2009;Nabetani and Ishikawa 2009;Min et al 2017).…”

“…APBs consist of a PML and Sp100 shell bound together by SUMO-SIM interactions and contain, in addition to telomeric DNA, the long non-coding RNA (lncRNA) telomeric repeat-containing RNA (TERRA), telomere associated proteins and several DNA damage factors (Yeager et al 1999;Arora et al 2014). APBs have been proposed to play a critical role in ALT by clustering telomeres and DNA repair factors together, thus concentrating substrates and enzymes required for recombination-based telomere elongation (Henson et al 2002;Cesare and Reddel 2010;Chung et al 2011;Chung et al 2012;Min et al 2019;Verma et al 2019). In agreement with this hypothesis, it has recently been shown that telomeres can be elongated during mitosis in APB-like foci in a process termed mitotic DNA synthesis MiDAS (Ozer et al 2018;Min et al 2019).…”