Telomeres: protecting chromosomes against genome instability

Abstract

The natural ends of linear chromosomes require unique genetic and structural adaptations to facilitate the protection of genetic material. This is achieved by the sequestration of the telomeric sequence into a protective nucleoprotein cap that masks the ends from constitutive exposure to the DNA damage response machinery. When telomeres are unmasked, genome instability arises. Balancing capping requirements with telomere replication and the enzymatic processing steps that are obligatory for telomere function is a complex problem. Telomeric proteins and their interacting factors create an environment at chromosome ends that inhibits DNA repair; however, the repair machinery is essential for proper telomere function.

Figure 1. The structure of human telomeres

Human telomeres consist of many kilobases of TTAGGG repeats, with a G rich leading strand and a C rich lagging strand. The G strand extends in the 3’ direction, forming the G-tail. The shelterin complex , consisting of the double stranded telomeric repeat binding factors TRF1 and TRF2, the TRF2 interacting factor RAP1, the bridging molecules TIN2 and TPP1 and the telomeric protection factor POT1, together covering the double and single stranded repeats. Shelterin members interact with a large number of other factors that transiently localize to telomeres, frequently in a cell-cycle dependent manner. These factors aid in the generation of a protective structure at chromosome ends, here referred to as telomeric loop, or T-loop. The T loop is generated by invasion of the single stranded G-overhang into the double stranded TTAGGG repeats. The looped structure protects telomeres on several levels. Invasion effectively sequesters the G-tail, and allows distinction of natural chromosome ends from double stranded breaks. The ATM dependent signaling cascade is inhibited by TRF2 and the ATR signaling pathway by POT1. Telomerase is likely inhibited by the complex, and it is suspected that TERRA play a role in this inhibition.

Figure 2. (A) Damage Sensing Pathways

In the event of single stranded breaks or fork stalling ATR is activated and RPA (Replication Protein A) binds to the exposed strands. ATR then phosphorylates RAD17, the 9-1-1 complex and TopBP1 (Topoisomerase IIβ Binding Protein 1), as well as CHK1 which amplifies the signal and mediates cell cycle arrest via Cdc25a. At double strand breaks (DSBs) the chromatin structure surrounding the break is dynamically re-structured, exemplified by ATM dependent phosphorylation of H2AX and modification of adjacent chromatin. The sensing of the DSB by the MRN complex triggers targeting of downstream mediators and activation of DNA repair pathways. The key event is the ATM dependent activation of CHK2 and p53, inducing arrest in G1 and G2/M phases of the cell cycle. Failure to repair results in permanent cell cycle arrest, senescence or apoptosis. (B) DNA Repair Pathways. The two primary pathways for the DNA repair are NHEJ and HR. NHEJ is the major pathway as it functions throughout the cell cycle. NHEJ requires sensing of the lesion by Ku70/80, activation of the DNA-PK (DNA dependent Protein Kinase) complex and 3’-5’ endonucleolytic resection of the break site. The break is then filled in by DNA Polymerases υ and λ and the repaired ends are fused by DNA Ligase IV. However, NHEJ is error-prone and defects in NHEJ are frequently linked with cancer. If DNA is damaged during S-phase the cell employs the error free HR pathway. ATM and MRN mediate recognition and resection of the break, the ssDNA overhang is detected by ATR and RPA, which promote association of RAD51/RAD52. The HR machinery mediates the synthesis of a new DNA strand using the overhang sequence as template. This mechanism ensures that the original DNA sequence can be faithfully restored and genetic integrity is maintained.

Figure 3. Telomeres as the cause of genome instability

When telomeres lose protection, either due to extensive loss of TTAGGG repeats or due to loss of protective factors such as TRF2 and POT1, they are recognized as damage in a pathway that depends on the MRN complex and the AT kinase. Next the G overhang is lost, then the chromatin structure changes and 53BP1 is recruited to allow for greater mobility, which facilitates NHEJ dependent covalent fusion of chromosome ends. In the absence of the p53 and pRb dependent tumor suppressor pathways cells containing nuclei with fused chromosome ends continue to cycle. When the fused chromosomes pass through mitosis they break randomly, leading to unequal distribution of genetic material in the daughter cells. These fusion-breakage-bridge cycles continue through the following cell divisions, leading to multiple non-reciprocal translocations and genome instability.

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