10-15% of cancers utilise a homology-directed repair mechanism known as Alternative Lengthening of Telomeres (ALT) to maintain their telomeres. Cancers engaging in ALT are typically aggressive with poor prognosis. While many aspects of the ALT mechanism have been characterised, the cell cycle regulation of telomere synthesis remains to be elucidated.
We identify for the first time, that ALT cells engage in two modes of telomere synthesis. First, non-productive telomere synthesis, which is defined as the generation of newly synthesised extrachromosomal telomeric repeat (ECTR) DNA in the absence of telomere lengthening, and second, productive telomere synthesis, which is defined as nascent telomere extension and telomere length maintenance. We demonstrate that ALT cells arrested in G2-phase synthesise substantial amounts of telomeric DNA. However, rather than the telomeres being extended, substantial amounts of newly synthesised telomeric DNA are then released into the cytoplasm during M-phase, in the form of ECTRs, coinciding with the inability of the cell to retain G2 nascent telomere synthesis into the subsequent G1-phase.
Contrary to the current dogma that ALT telomere lengthening occurs in both G2 and M-phases of the cell-cycle, we show that productive telomere synthesis or extension occurs specifically during prometaphase. We demonstrate that most factors involved in break-induced telomere synthesis can be grouped into affecting both productive and non-productive telomere synthesis in ALT. However, we show that RAD51 and POLH specifically promote productive telomere lengthening, and protect against telomere dysfunction and associated non-productive telomere synthesis. This has major implications for the therapeutic targeting of ALT cancers via cell-cycle disruption and targeting of telomere-associated DNA repair factors.