Linear chromosomes wear a snugly fitting nucleoprotein cap, which prevents degradation of the chromosome ends and protects against inappropriate recombination. In mammalian cells, this cap — the telomere — comprises a repetitive G-rich sequence bound by a number of proteins, including Ku70, Ku80, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and the telomeric-repeat binding factor 2 (TRF2). Goodwin and colleagues have now investigated how capping occurs and, reporting in Science, they discuss how the processing of telomeres is linked to their mode of replication.

Two TCCs in HTC75 cells. The tel-G probe (which detects leading-strand telomeres) was hybridized and photographed, then the probe removed by denaturation and the tel-C probe hybridized and photographed. Yellow arrowheads indicate the point of fusion. Image courtesy of Michael Cornforth, University of Texas. (Cen, centromere.)

To investigate the capping mechanism, the authors used a cell line containing a dominant-negative mutant of TRF2 (TRF2ΔBΔM), which removes endogenous TRF2 from telomeres. They expressed TRF2ΔBΔM for five days in HTC75 human fibrosarcoma cells, and found that 44 of 154 mitotic cells showed end-to-end chromosomal fusions. These fusions — dubbed telomeric chromatid concatenates (TCCs) by the authors — involved just one sister chromatid from each of the two fusing chromosomes (see image), indicating that fusion, and hence TRF2 capping, must have occurred after telomere replication.

Telomere replication poses special challenges. The cap must not only disassemble for replication to occur, but it must also re-form afterwards. Replication involves the generation of two new telomeres — one produced by leading-strand DNA synthesis, the other through lagging-strand synthesis.

To study this process further, Goodwin and colleagues used a technique known as chromosome-orientation fluorescence in situ hybridization, which produces different hybridization patterns depending on the type of fusion that has occurred — that is, lagging–lagging, lagging–leading or leading–leading strand fusions. The authors then asked whether the impaired capping seen with TRF2ΔBΔM is limited to telomeres synthesized by one mode of replication, or whether it is a random process. They found that, in 133 out of 135 cases, TCCs were produced by fusion between leading-strand telomeres.

Why does capping fail to occur only on the leading-strand telomeres? Goodwin and colleagues speculate that this might be due to the ends generated by the two modes of replication — leading strands are blunt ended, so have an absolute requirement for TRF2 and DNA-PKcs to fashion 3′ overhangs prior to the formation of a t-loop at the chromosome end. Lagging-strand telomeres, on the other hand, already have a 3′ overhang after replication. Alternatively, they say, there may be other differences in how the two types of telomere are capped.