Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination

Telomerase-independent ALT (alternative lengthening of telomeres) cells are characterized by high frequency of telomeric homologous recombination (HR), C-rich extrachromosomal circles (C-circles) and C-rich terminal 5' overhangs (C-overhangs). However, underlying mechanism is poorly understood. Here, we show that both C-circle and C-overhang form when replication fork collapse is induced by strand break at telomeres. We find that endogenous DNA break predominantly occur on C-rich strand of telomeres in ALT cells, resulting in high frequency of replication fork collapse. While collapsed forks could be rescued by replication fork regression leading to telomeric homologous recombination, those unresolved are converted to C-circles and C-overhang at lagging and leading synthesized strand, respectively. Meanwhile, multiple hallmarks of ALT are provoked, suggesting that strand break-induced replication stress underlies ALT. These findings provide a molecular basis underlying telomeric HR and biogenesis of C-circle and C-overhang, thus implicating the specific mechanism to resolve strand break-induced replication defect at telomeres in ALT cells. Author summary 10 to 15% human cancers utilize telomerase-independent alternative lengthening of telomeres (ALT) to maintain their telomere length. Unexpectedly, we find that endogenous C-strand breaks predominantly exist in telomeres of ALT cells, which induce high frequency of replication fork collapse. While collapsed fork triggers fork regression machinery to restart the replication, leading to telomeric homologous recombination; those unresolved are converted to C-circle and C-overhang. These findings suggest that the formation of C-circle and C-overhang represents a unique manner for ALT cells to prevent chromosome instability induced by replication defect at telomeres. Moreover, multiple hallmarks of ALT are provoked during this process, demonstrating that DNA strand break at telomeres underlies ALT mechanism.