The evolutionary transition from circular to linear chromosomes brought with it two new challenges to genome stability. The first of these, known as the “end replication problem”, relates to the loss of nucleotides from the 5′ terminus of the lagging strand after every round of DNA replication. The second challenge to genome stability associated with linear chromosomes is that of preventing the chromosome termini from being recognized and processed as DSBs. The specialized repetitive sequences and protein-DNA complexes that comprise telomeres function both to maintain the chromosome ends and to provide protection from the DNA repair machinery.
Removal of the RNA primers from lagging strand Okazaki fragments results in a gap of missing nucleotides at the 5′ terminus, which cannot be filled by DNA polymerases due to the strict 5′ → 3′ polarity of their synthesis activity. Consequently, in the absence of a prophylactic mechanism, continuous cycles of replication would cause the genomes of individual organisms to grow progressively shorter and genes would be lost [140
]. The addition of telomeres, non-coding repetitive DNA sequences, to the termini of chromosomes overcomes this problem. Telomerase, a specialized reverse transcriptase that carries its own RNA template, synthesizes telomeres of sufficient size to ensure that genetic information is not lost due to the end replication problem. After telomerase has synthesized a guanine-rich 3′ single stranded extension (composed of TTAGGG repeats in humans) at the chromosome terminus, the cytosine-rich complementary strand is synthesized by traditional semi-conservative replication. In yeast, telomerase is active at each round of DNA replication, thereby ensuring that telomere length maintains a steady state. In contrast, during vertebrate development and within stem cell populations, telomerase synthesizes telomeres with a length that is sufficient to sustain numerous future replication cycles. Since telomerase is inactive in somatic cells, telomeres grow progressively shorter over time until they reach a critical length, at which point cell senescence or apoptosis is triggered. Vertebrate telomeres therefore provide a solution to the end replication problem in a temporally finite manner. This brings the added benefit of tumor suppression, in that unchecked cellular replication associated with cancer is thwarted when these cells reach their critical telomere length.
Throughout Eukarya, telomeres are bound by specific proteins that sequester the free chromosome termini within a nucleoprotein cap [141
]. In higher eukaryotes these same telomere-binding proteins additionally promote the formation of a unique lariat-like structure called the “t-loop”, which provides a second layer of protection from the DSB repair machinery [140
]. Critical to t-loop formation is the generation of a 3′ single-stranded overhang (termed the “G-overhang” because it is present on the G-rich strand of the telomere) at each chromosome terminus. Since the newly synthesized lagging strand is missing a portion of its 5′ terminus, a short 3′ G-overhang (~10 nucleotides) is inherently present at this end of a newly synthesized chromosome. In contrast, leading strand synthesis generates a blunt chromosome end. In higher eukaryotes both this blunt end and the short G-overhang at the opposite chromosome end are processed by an unknown nuclease to generate mature G-overhangs 50–300 nucleotides in length [140
]. Folding each chromosome terminus back upon itself enables the G-overhang to invade and base pair with the complementary strand (analogous to what occurs during HR), giving rise to the t-loop lariat (). Although G-overhangs are also present at telomeres in S. cerevisiae
, they are only 12–14 nucleotides in length [143
] and t-loops have not been observed. Regardless of whether or not t-loops are employed, the importance of sequestering telomeres into nucleoprotein caps is made clear by the fact that cap disruption can result in shortening or lengthening of telomeres, telomere fusion, telomere loss, elevated levels of recombination, and checkpoint activation [144
Human telomere structure
In vertebrates stabilization of telomeres and formation of the t-loop are facilitated by the shelterin complex, which consists of the following six proteins: Telomeric Repeat-binding Factors 1 and 2 (TRF1 and TRF2), repressor and activator protein 1 (RAP1), TRF1-interacting nuclear protein 2 (TIN2), protection of telomeres 1 (POT1), and TIN2- and POT1-interacting protein (TPP1) [141
]. Whereas TRF1 and TRF2 bind the double-stranded region of the telomere, POT1 has affinity specifically for the single-stranded G-overhang (). TIN2 bridges TRF1 and TRF2, while TPP1 bridges TIN2 and POT1. RAP1 is recruited to telomeres via its interaction with TRF2 [140
]. Details regarding the vertebrate shelterin complex, its homologs in other organisms, and other facets of telomere structure and function are examined elsewhere in this issue.
The importance of the MRX complex for normal telomere maintenance was first recognized many years ago when deletion or disruption of S. cerevisiae
], Mre11, or Xrs2 [146
] were reported to result in shortened telomeres and cell senescence [147
]. Lundblad and coworkers subsequently demonstrated MRX to be in the same S. cerevisiae
epistasis group as telomerase [148
]. Consistent with this notion, S. cerevisiae
MRX associates with telomeres in late S-phase [149
] when yeast telomeres are synthesized, and in the absence of functional MRX the single-stranded telomeric DNA binding protein Cdc13 is unable to bind to telomeres [149
]. Multiple groups have independently demonstrated that in both S. cerevisiae
and S. pombe
telomere length [151
] and the specific formation of G-overhangs [149
] are unaffected by the nuclease-inactivating D56N or H125N mutations of Mre11. Moreover, telomerase-mediated replication of telomeres is defective in Mre11 null cells but not in the Mre11-D56N or Mre11-H125N backgrounds [153
]. Targeting S. cerevisiae
telomerase to telomeres via fusion with Cdc13 overcomes telomere maintenance defects resulting from a non-functional MRX complex [153
]. Collectively, these observations suggest that MRX facilitates the recruitment of telomerase to telomeres. Consistent with this, in S. cerevisiae
the specific recruitment of telomerase to telomeres during the S phase is abolished by the absence of Mre11 [149
]. While the MRX-mediated recruitment of yeast telomerase to telomeres could involve a direct interaction between these proteins, it could also simply be a consequence of MRX promoting the processing of telomeres into a form suitable for telomerase sequestration. Considering the well established role of MRX as a promoter of 5′ → 3′ resection at DSBs, the specificity of Cdc13 for ssDNA, and the affinity of Cdc13 for telomerase, it is tempting to speculate that MRX works with Sae2 to generate short 3′ overhangs that are bound by Cdc13 and subsequently recruit telomerase.
The first indication that MRN may also contribute to telomere maintenance in higher eukaryotes was the identification of Nbs1 and Mre11 sequestered at telomeres in meiotic human fibroblasts [154
]. It was subsequently shown that MRN specifically associates with the TRF2 component of shelterin, and that Nbs1 accumulates at telomeres in S phase but not during G1 or G2 [155
]. Since TRF2 does not associate with ionizing radiation induced DSBs, this interaction appears to occur exclusively within the telomere micro-environment.
Cultured human NBS fibroblasts display shortened telomeres. In these cells, the coexpression of Nbs1 along with telomerase results in longer telomeres than the expression of telomerase alone [156
]. This suggests that, analogous to the situation in yeast, human MRN facilitates telomerase activity at telomeres. Further supporting this notion, knockdown of Mre11 or Nbs1 in cultured human cancer cells specifically reduces the length of 3′ G-overhangs, but has no effect in cells that do not express telomerase [157
]. Collectively, these data demonstrate that MRN facilitates the action of telomerase at chromosome termini in mammalian cells. Whether MRN facilitates telomerase activity by modifying telomere ends, opening up the t-loop, altering chromatin structure, or by directly associating with telomerase remains to be determined. In telomerase-negative primary human cells, chromatin immuno-precipitation revealed that Mre11, phosphorylated Nbs1, and ATM were bound to telomeres in the G2 phase of the cell cycle [158
]. This study suggested that telomeres become accessible in G2 and are recognized as DNA damage. A localized DNA damage response at telomeres may therefore be required for recruiting the processing machinery that is responsible for formation of the end protection complex.
Telomeres can be rendered dysfunctional by removal of the telomere DNA binding protein TRF2 from the shelterin complex. These “uncapped” telomeres are recognized as DSBs, and result in ATM activation, phosphorylation of Chk2 and H2AX, the formation of 53BP1-associated telomere-induced DNA damage foci (TIF), and NHEJ-mediated chromosome fusion [159
]. The role of MRN at dysfunctional telomeres has recently been studied in embryonic fibroblasts derived from mice harboring MRN mutations and deletions, combined with TRF2 removal by conditional deletion or shRNA knockdown [160
]. These studies demonstrate that MRN is required for ATM signaling in response to telomere dysfunction. When TRF2 levels are depleted by shRNA, ATM activation and TIF formation are reduced in the Mre11Δ/Δ
null background but remain robust in the nuclease deficient Mre11H129N/Δ
]. This suggests that the MRN complex functions to detect and signal the presence of dysfunctional uncapped telomeres, and that this capability does not depend on the Mre11 nuclease activity.
Studies of dysfunctional telomeres performed in TRF2 deficient cells, have revealed a complex role for MRN in telomere fusion by NHEJ. Depending on the stage of the cell cycle and the specific structure of the telomere terminus, MRN can either promote or suppress the NHEJ-mediated fusion of dysfunctional telomeres. Conditional double-knockout of Nbs1 and TRF2 results in abrogated fusions in G1, due to defects in ATM-dependent signaling [161
]. Although in G1 the MRN complex may promote NHEJ, the role of MRN after replication in G2 appears to be very different [161
], and here the ability of MRN to promote end-to-end chromosome fusions at uncapped telomeres depends on Mre11’s nuclease activity [162
]. In another study employing TRF2 knockdown cells, the number of chromosome fusions in the Mre11Δ/Δ
backgrounds was 15-fold lower than that observed in an Mre11 active background [162
]. To test whether Mre11 promotes NHEJ of TRF2-uncapped telomeres by removing the 3′ G-overhang, an in-gel hybridization assay was employed. In contrast to cells with functional Mre11 where the 3′ overhang is rapidly degraded, in Mre11Δ/Δ
cells the overhang persists [162
]. This suggests that the Mre11 nuclease activity is required for processing of 3′ overhangs to allow efficient NHEJ of telomere ends when rendered dysfunctional by TRF2 removal. An especially illuminating finding of these TRF2-uncapped telomere studies was that even though they occurred much less frequently, telomere-telomere fusions still occurred in the absence of functional MRN [160
]. Importantly, the majority (~90%) of these residual telomere fusions involved the leading strands of sister chromatids. In the Mre11H129N/Δ
background only ~60% of telomere-telomere fusions involved the leading strands of sister chromatids [162
]. The authors suggested that this can be explained by the structural differences between telomere termini generated by leading versus lagging-strand replication. Leading-strand replication generates a blunt-ended telomere terminus that can readily be fused via NHEJ without prior nuclease processing. In contrast, lagging-strand replication generates a 3′ telomeric overhang, which is incompatible with DNA ligation [159
] and would therefore require nuclease processing prior to fusion. Thus, within a TRF2 deficient background the MRN complex appears to prevent
the fusion of newly replicated leading strand telomeres by promoting 5′ end resection to give NHEJ-incompatible 3′ overhangs. It will be interesting to determine the extent to which CtIP and other end-processing factors are involved here.
The cellular response to dysfunctional telomeres is in many ways similar to the response induced by non-telomeric DSBs. In both of these contexts MRN is required for ATM activation, and many of the factors that accumulate at ionizing radiation-induced foci (IRIF) also accumulate at TIF. Moreover, both damage-induced DSBs and dysfunctional telomeres can lead to the same signaling pathways, cell cycle arrest, and apoptosis [163
]. Differences between damage-induced DSBs and uncapped telomeres include (i
) the fact that TRF2 suppresses ATM activation only within the telomere microenvironment since it is abundant at chromosome ends but not elsewhere in the nucleus [165
], and (ii
) DNA processing is not required for the ATM-mediated damage response at telomeres [159