Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase reverse transcriptase, telomerase RNA, and dyskerin.

Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.