Human telomeres are bound by two major double-strand telomere binding proteins, TRF1 and TRF2. Both proteins were identified on the basis of their ability to bind human telomeric oligonucleotides in vitro (Chong et al., 1995; Broccoli et al., 1997). As with most other double-strand telomere binding proteins, TRF1 and TRF2 contain a Myb-like helix-turn-helix DNA-binding domain at their C-terminus. TRF1 and TRF2 proteins share 56% identity in their Myb domain. The central dimerization domain is responsible for the formation of TRF1 and TRF2 homodimers (Bilaud et al., 1997). The most variable region of the proteins is the N-terminal domain, which is acidic in TRF1
and basic in TRF2. Both proteins specifically bind telomeric DNA in vivo (Broccoli et al., 1997; Bilaud et al., 1997). TRF1 dimer binds adjacent YTAGGGTTR sites on the DNA. The bound TRF1 dimers loop the sequence between the two half sites (Bianchi et al., 1997) and also have the ability to bring two telomeric tracts together (Griffith et al., 1998). Both TRF1 and TRF2 are extremely important for telomere length regulation (van Steensel and de Lange, 1997; Smogorzewska et al., 2000). Over-expression of TRF1 and TRF2 in human cell lines results in a gradual decline in telomere length, whereas over- expression of a dominant negative form of TRF1 leads to telomere elongation. These studies suggest that telomere-bound TRF1 is an inhibitor of telomere elongation.
One of the many important roles of TRF1 proteins at the telomere is the ability to interact with other telomere proteins, such as TIN2, Ku and tankyrase. The function of the conserved Ku heterodimer at the telomeres will be discussed below. Tankyrase is a poly (ADP-ribose) polymerase, containing 24 typical ankyrin repeats and an N-terminal acidic domain. The human tankyrase promotes telomere elongation by modifying TRF1 and inhibiting its binding to telomeric DNA (Smith et al., 1998). Over-expression of tankyrase induces ADP ribosylation of TRF1, releasing it from the telomere. Subsequently, the lack of a sufficient number of bound TRF1 molecules leads to gradual telomere elongation by telomerase.
Another telomeric protein, TIN2, interacts with the central domain of TRF1 and co-localizes with TRF1 at telomeres (Kim et al., 1999b). Over-expression of a dominant- negative TIN2 lacking a portion of its amino-terminal sequence induces telomere elongation only in telomerase-positive, but not telomerase-negative human cells, suggesting that TIN2 may also be a negative regulator of telomere elongation. It has been
promoting the formation of a large, multimeric complex at the telomeres (Kim et al., 1999b).
Recently, a novel TRF1-interacting protein called PinX1 was identified in a yeast two-hybrid screen (Zhou and Lu, 2001). Over-expression of PinX1 induces telomere shortening, leading to genome instability. PinX1 depletion results in elongation of telomeres. Remarkably, PinX1 was shown to inhibit telomerase activity both in vitro and in vivo by direct interaction with hTERT. Therefore, PinX1 is the first known telomerase inhibitor in vivo.
The second mammalian duplex telomere protein is TRF2. This protein binds along the length of the double-stranded telomere region with more than 100 protein molecules per chromosome end (Bilaud et al., 1997; Broccoli et al., 1997). Like TRF1 and TIN2, TRF2 is a negative regulator of telomere length (Smogorzewska et al., 2000). In recent years, TRF2 has also emerged as the major telomere end protection factor. Over-expression of a dominant-negative version of TRF2 results in the disappearance of endogenous TRF2 from telomeres, induction of p53-damage response pathway and chromosome end fusions (van Steensel et al., 1998; Karlseder et al., 1999). The end-to- end telomere fusions result from loss of the G-overhang. The G-overhang is an essential feature of functional telomeres, and its absence results in telomeres being recognized as double-strand DNA breaks. The cellular machinery responsible for repairing double- strand DNA breaks in higher eukaryotes is a multisubunit protein complex associated with the NHEJ pathway. This pathway is activated in cells lacking TRF2 and is thought to be responsible for fusing telomere ends in these mutants. In some cell types, TRF2
inhibition also induces apoptosis (Karlseder et al., 1999). Therefore, the loss of TRF2 from telomeres creates an altered chromosome end structure capable of activating the DNA damage response pathway and ultimately resulting in senescence or apoptosis.
TRF2 also protects critically shortened telomeres. In culture, primary human lung fibroblasts grow for 55 population doublings before reaching senescence with an average telomere length of 7 kb (Karlseder et al., 2002). Over-expression of wild-type TRF2 allows these cells to divide longer, with telomere length at senescence reaching only 4 kb (Karlseder et al., 2002). These cells also accumulate fewer chromosomal aberrations, suggesting that high levels of TRF2 protein provide better protection of chromosome ends. The extra stability provided by TRF2 over-expression could result from more efficient t-loop formation (Griffith et al., 1999). The current model suggests that in addition to TRF2’s role in the inhibition of NHEJ processes at the natural chromosomal termini, this protein is also involved in chromosome end processing and overhang formation, which in turn are necessary to form stable t-loops (Karlseder, 2003; de Lange, 2002).
TRF2 also interacts with hRap1 and recruits it to telomeres. hRap1 protein was identified in a standard two-hybrid screen using TRF2 as bait (Li et al., 2000). Based on the sequence similarity, hRap1 was classified as a human ortholog of the Saccharomyces cerevisiae telomere protein Rap1. Human Rap1 is a 399 amino acid protein and contains significant sequence identity (24-25%) to three different domains in the yeast Rap1p. As in yeast, hRap1 harbors an N-terminal BRCT domain, but contains only one central DNA-binding Myb domain. Consistent with the data in yeast, human Rap1 is concentrated at telomeres throughout the cell cycle. Although hRap1 does not bind
expression of full-length hRap1 leads to a gradual telomere shortening, consistent with its role as a negative regulator of telomere length (Li et al., 2000).
DNA DAMAGE RESPONSE PROTEINS AS IMPORTANT COMPONENTS OF