In the 1930s, pioneering work by Muller with flies and by McClintock with maize [103
] led to the description of telomeres as structures that protect chromosomes from loss and end-to-end fusions. Telomere replication probably requires the transient loss of protective protein–DNA structure(s) to allow the replication machinery access to DNA. For telomeres organized in T-loops, passage of the replication fork would seem to demand a transient loss of the T-loop.
One possibility to explain the presence of checkpoint and repair proteins at telomeres is that, during their replication, telomeres are seen as DSBs. This view is supported by the fact that, soon after semi-conservative DNA replication, the C-strand of S. cerevisiae
telomeres is specifically degraded [16
], a clear loss of the normal role of the telomere in protecting ends from degradation. Telomeres in human fibroblasts that do not express telomerase also appear to lose their protective structure late in the cell cycle, because they become substrates for labeling by terminal transferase [92
]. Given that this access to DNA-modifying activities occurs even in asynchronous cells, it cannot be attributed to aphidicolin-induced telomere damage. Nonetheless, if replicating telomeres are seen transiently as DSBs, their semi-conservative replication cannot elicit a classic checkpoint-mediated cell cycle arrest, because it happens at every telomere in every S phase.
Another possibility is that telomerase-mediated lengthening, which occurs at only a subset of telomeres, creates a structure that is seen as a DSB. Consistent with this view, the S. cerevisiae
proteins that bind to DSBs are also crucial for telomerase-mediated lengthening and bind preferentially to the short telomeres that are the preferred substrates for telomerase. As discussed above, the order of appearance of these proteins at the telomere (MRX followed by Tel1), their genetic dependencies (binding of Tel1 requires the carboxyl end of Xrs2), and their consequences (MRX-promoted degradation of the 5′ strand) are identical to the early steps in DSB processing ().However, in the inducible short telomere system, the presence of a single short telomere does not affect cell cycle progression, as monitored by the timing of cell cycle landmarks, such as RNR1
RNA or cyclin Clb2 expression [77
]. Likewise, STEX demonstrates that ~10% of wild type telomeres are lengthened by telomerase in a given cell cycle [72
]. The most plausible scenario is that these lengthening telomeres are distributed among most cells in the population, rather than being concentrated in a small fraction of cells. If so, given that a haploid S. cerevisiae
cell has 32 telomeres, cells must tolerate at least a few telomeres undergoing telomerase lengthening without triggering a checkpoint-mediated cell cycle arrest. However, the DNA damage checkpoint is activated if most telomeres in the cell are undergoing telomerase lengthening, as occurs as a result of sudden Tel1 overexpression in cells in which all telomeres are initially very short [93
]. Even a single elongating S. cerevisiae
telomere might provoke a subset of checkpoint-mediated downstream events (e.g. phosphorylation of the Rad53 checkpoint kinase [94
]) and yet not trigger cell cycle arrest. In this respect, the response to a few short telomeres might be similar to what occurs in response to stalled replication forks during the S. cerevisiae
S phase. A threshold number of stalled forks is needed to elicit an intra-S phase checkpoint, but a smaller number can cause a sub-threshold level of Rad53 phosphorylation [95
]. Likewise, reducing POT1 levels in human tumor cells results in G-tail shortening and a transient DNA damage response but not a cell cycle arrest [98
So how are telomeres distinguished from DSBs during their preparation for telomerase elongation? To date, the data providing an answer to this question come mainly from S. cerevisiae
, in which the early events in telomerase lengthening are indistinguishable from what occurs at DSBs. MRX and Tel1 are recruited to both short telomeres and to DSBs (). However, the subsequent binding of Mec1p is readily detected at DSBs [6
] but not at short telomeres (M. Sabourin and V.A. Zakian, unpublished). Moreover, when a DSB is generated near a tract of telomeric sequence that has been inserted at an internal site on a chromosome, both the binding of Mec1 to the break and the subsequent cell cycle arrest are abbreviated [99
]. Given that the checkpoint response to DSBs is Mec1-dependent, the absence of Mec1 from elongating telomeres is probably sufficient to explain why telomerase action does not impede cell cycle progression.
MRX-promoted degradation of the 5′ strand at a DSB generates 3′ single-stranded DNA with a sequence that is determined by the site of the break. However, at telomeres, 5′ strand degradation always generates G-strand telomeric DNA. The Cdc13 and POT1 complexes bind specifically to these G-tails, whereas RPA binds non-specifically to single-stranded DNA at DSBs. RPA then recruits Mec1, but this does not occur at S. cerevisiae
telomeres, presumably because the Cdc13 complex does not interact with Mec1p. Cdc13 also limits the extent of C-strand degradation, preventing it from reaching non-telomeric sequences. Reducing Cdc13 function by, for example, growing cdc13–1
cells at high temperatures, leads to unchecked C-strand degradation and activation of a checkpoint-mediated arrest [10
]. Checkpoint activation in this context is due to the exposure of non-telomeric single-stranded DNA that can recruit RPA and Mec1. Thus, the binding of G-strand-specific binding proteins, such as Cdc13 and POT1, to the single-stranded DNA generated at telomeres – as opposed to binding of RPA to the single-stranded DNA exposed at a DSB – is probably the key event that distinguishes replicating telomeres from damaged DNA.