Alternative lengthening of telomeres (ALT) is a telomerase independent telomere 
maintenance mechanism that occurs in ∼15% of cancers. The potential mechanism of 
ALT is homology-directed telomere synthesis, but molecular mechanisms of how ALT 
maintains telomere length in human cancer is poorly understood. Here, we 
generated TERC (telomerase RNA) gene knockouts in telomerase positive cell lines 
that resulted in long-term surviving clones acquiring the ALT pathway but at a 
very low frequency. By comparing these ALT cells with parental telomerase 
positive cells, we observed that ALT cells possess excessively long telomeric 
overhangs derived from telomere elongation processes that mostly occur during S 
phase. ALT cells exhibited preferential elongation of the telomeric lagging 
strands, whereas telomerase positive cells exhibited similar elongation between 
leading and lagging strands. We propose that the ALT pathway preferentially 
occurs at telomeric lagging strands leading to heterogeneous telomere lengths 
observed in most ALT cancers.