Telomere Maintenance Telomerase Proteins

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 Telomere Maintenance Telomerase Proteins Background

Telomerase recruitment to telomeres

The association of telomerase to chromosome ends has been shown to be cell-cycle regulated in mammals, fission yeast, and budding yeast. Moreover, telomerase has been shown to preferentially elongate short telomeres. The regulation of telomere length by telomerase has been linked to the telomeric protein/DNA complexes, bound to the dsDNA repeats and ssDNA region. The process has been identified as "protein counting" model for telomerase regulation. In this model, Bianchi and Shore suggest that when a significant number of negative regulators of telomere length are bound to the double-stranded telomeric region, telomerase recruitment is inhibited. However, following several rounds of cell division and telomere repeat loss, the number of telomerase inhibitors drops which reverses the inhibition of telomerase, and leads to the addition of telomere repeats. This process is thought to potentially involve members of the shelterin complex, which will be described in the next section.


Telomere-associated proteins: Maintenance and Protection

The shelterin complex

The telomere region has been shown to serve as a platform for a myriad of proteins, ranging from repair and checkpoint proteins, to the shelterin complex which plays a protective as well as a replication regulator role. The subtle regulation of these proteins' modifications in addition to the timing and stability of their binding is crucial to ensure proper telomere maintenance. The human shelterin complex includes the proteins TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, several of which have been shown to have fission yeast orthologues or functional homologues.

Rap1, which is related to the mammalian and budding yeast Rap1, does not bind directly to telomeric DNA but is recruited to telomeres via its interaction with Taz1. This is in contrast with the situation in budding yeast, in which Rap1 binds directly to telomeric DNA. In mammals, much like in fission yeast, Rap1 is dependent on TRF2 for telomere binding and its deletion affects the length of mammalian telomeres as well.

Poz1, the functional counterpart of mammalian TIN2, plays a central position in the shelterin complex, since it connects Taz1 to the G-tail binding protein Pot1 by simultaneously interacting with Rap1 and the Pot1 interacting partner Tpz1. Much like taz1 and rap1, the deletion of poz1 causes massive telomerase-dependent expansion of the G-rich region, thus implicating it in the negative regulation of telomerase activity.

Tpz1, the fission yeast counterpart of TPP1, interacts with Pot1 as well as Poz1 and Ccq1, which makes it the central protein necessary for the formation of the Pot1 subcomplex, comprising Pot1, Tpz1, Ccq1 and Poz1. Poz1 and Ccq1 have been shown to be redundantly required for the telomere protection function of Pot1 and Tpz1.

Pot1, (protection of telomeres 1) is an end-capping protein identified in a wide variety of species. Fission yeast Pot1 is crucial for telomere maintenance, and human POT1 has been implicated in telomere regulation. Telomere protection by POT1 in mammals has been shown to require its interaction with TPP1 (Tpz1 homologue). The N-terminal oligonucleotide/oligosaccharide- binding (OB) fold domain of fission yeast Pot1 enables it to specifically bind the ssDNA telomeric region. The deletion of fission yeast Pot1, much like its mammalian counterpart, leads to telomere elongation, but its overexpression seems to have the same effect indicating that Pot1 affects telomerase both positively and negatively.

In addition to the proteins described above, which have corresponding counterparts in the mammalian shelterin complex, the fission yeast shelterin-like complex has been shown to interact with one additional protein, Ccq1. Ccq1 is required for the recruitment of telomerase to telomeres and the inhibition of recombination at telomeres. Ccq1 was also identified as a member of a multi-enzyme complex SHREC (Snf2/Hdac-containing Repressor Complex) and has been shown to contribute to the formation of telomeric heterochromatin. It is possible that the mammalian shelterin complex might interact with a Ccq1-like protein that remains to be discovered.


Shelterin and DNA damage

One of the main functions of telomeres is to prevent the activation of a DNA damage response. Different components of the mammalian shelterin complex, namely TRF2 and POT1, can repress DNA damage signaling by inhibiting the recruitment of the ATM or ATR kinases respectively. Ataxia telangiectasia and Rad3 related (ATR) and Ataxia telangiectasia mutated (ATM) are serine/threonine-specific kinases that are involved in sensing DNA damage and activating the DNA damage response, leading to cell cycle arrest. ATR is activated in response to persistent single-stranded DNA, which is a common intermediate formed during DNA damage detection and repair. When TRF2 is eliminated from the cells, the telomeres are treated as double-strand breaks (DSBs). The ATM/MRN complex is then recruited to the telomeres and strongly activates the downstream targets p53 and Chk2 by phosphorylating them. This leads to the inhibition of cell cycle progression via the activation of p21 and the inhibition of Ccd25. On the other hand, when POT1 is lost from the cells, the consequence at the telomeres is the same as the one that follows replication fork stalling and the occurrence of ssDNA, whereby ATR/ATRIP get recruited to the site and activate downstream p53 and Chk1 via phosphorylation. In turn, cell cycle progression is inhibited as is the case for the ATM/MRN regulated pathway. Whether it is TRF2 or POT1 that is lost, 53BP1, MDC1 and H2AX are recruited to the telomere, and form telomere-induced foci (TIFs) that are similar to the DNA-damage foci at DSBs. These foci can arise in all the stages of interphase and at all telomeres.

If telomeres are not protected by shelterin, they are identified as DSBs and are targeted to be repaired by NHEJ, which may lead to chromosome end fusions. While TRF2 seems to play an important role in the inhibition of NHEJ, POT1 only has a minor role.


Shelterin and telomerase recruitment

In budding yeast, which does not have a shelterin complex, telomerase is preferentially recruited to short telomeres. In a model presented by Bianchi and Shore, the MRX complex (Mrell, Rad50, Xrs2) is loaded to shortening telomeres, which leads to an Xrs2-dependent recruitment of Tell. Cdcl3 then gets phosphorylated by Tell, and such a modification allows it to interact with the telomerase subunit Estl and recruit the enzyme to the telomere ends.

In mammals, a similar scenario is proposed to be taking place as well, with some modifications. When in a t-loop configuration, which is formed by the invasion of the G-strand overhang of the double-stranded telomeric region, telomeres are inaccessible to telomerase. Furthermore, when the POT1-TPP1 subcomplex no longer associates with the TRF1-TRF2 portion of the complex and is found at the overhang, in an unfolded t-loop configuration, telomerase recruitment via its interaction with TPP1 ensues. This mechanism might be triggered by changes in telomere length, but direct evidence for this hypothesis is still underway.