Molecular characterization of inter-telomere and intra-telomere mutations in human ALT cells

Telomeres in most immortal cells 1 , 2 , 3 are maintained by the enzyme telomerase 4 , allowing cells to divide indefinitely. Some telomerase-negative tumors and immortal cell lines maintain long heterogeneous telomeres by the ALT (alternative lengthening of telomeres) mechanism 5 , 6 ; such tumors are expected to be resistant to anti-telomerase drug therapies. Occasionally telomerase-negative Saccharomyces cerevisiae mutants survive, and 10% of them (type II survivors) have unstable telomeres 7 , 8 . As in human ALT+ cells 9 , short telomeres in yeast type II survivors lengthen abruptly; in yeast, this is dependent on the recombination proteins Rad52p and Rad50p 10 . In human cells, ALT involves copying of sequence from a donor to a recipient telomere 11 . We have characterized for the first time a class of complex telomere mutations seen only in ALT+ cells. The mutant telomeres are defined by the replacement of the progenitor telomere at a discrete point (fusion point) with a different telomere repeat array. Among 19 characterized fusion points, one occurred within the first six repeats of the telomere, indicating that these recombination-like events can occur anywhere within the telomere. One mutant telomere may have been involved in a secondary recombination-like mutation event, suggesting that these mutations are sporadic but ongoing in ALT+ cells. We also identified simple intra-allelic mutations at high frequency, which evidently contribute to telomere instability in ALT+ cells.