End-Replication Problem and Telomerase

The semi-conservative nature of DNA replication prohibits DNA polymerase from replicating the 3‟-end of the lagging strand of the DNA (termed as the “end replication problem”) due to the absence of a RNA primer with a free 3‟-OH group. Therefore, after each round of cell division and DNA replication, the extreme 3‟-end of the telomeres shorten by about 50-150 base pairs and after about 60-70 cycles of cell division, the telomeres reach a critical length when the cell enters the senescence stage eventually leading to apoptosis and finally results in cell death[17d, 31d]_{. However, the telomere length is maintained at a constant}

level in tumor cell lines as well as in germ-line cells [29]_{. This is due to the activation of an}

enzyme called telomerase, which is absent for the normal function of most somatic cells that usually have longer telomeres, whereas widely expressed in immortal cells. In fact, telomeres are highly maintained in length in 80-85% of human tumor cells, which divide indefinitely by the action of telomerase[21d]_{. Telomerase is a ribonucleoprotein reverse-transcriptase enzyme}

consisting of an eleven bases long RNA template and a catalytic subunit (hTERT) with reverse transcriptase activity. With the aid of accessory proteins, the enzyme extends the 3‟ ends of the

DNA by consecutively adding the TTAGGG hexanucleotide repeats using the RNA template as a primer[21d]_{. However, in order to continue the elongation process, it is highly imperative that}

the primer maintain the single stranded conformation. Formation of any secondary structures, such as G-quadruplexes, impedes the hybridization of template RNA subunit of telomerase onto the primer during the elongation process and consequently resulting in the inhibition of telomerase activity. Therefore, inducing or stabilizing the telomeric quadruplex conformation might be one of the important methods of controlling and inhibiting the activity of telomerase. Moreover, the telomerase holoenzyme itself has potential recognition sites that can be exploited for the development of inhibitors. Antisense oligodeoxynucleotides[33]_{, hammerhead}

ribozymes[34]_{, peptide nucleic acids}[35]_{, chimeric RNA modules}[36]_{, reverse transcriptase}

inhibitors[37] _{and immunotherapy agents}[38] _{are some of the agents that are shown to}

potentially recognize different structural and functional units of telomerase holoenzyme, and are actively studied as telomerase inhibitors. Since telomerase is necessary for the immortality of many cancer types, it is thought to be a potentially highly selective and attractive drug target for several anti-tumour strategies. Its action is detected in most primary human tumor specimens and tumour-derived cell lines, such as those of the prostate, breast, colon, lung and liver [21c-e, 31c, 39]_{. Hence, inactivation of telomerase may play an important role in cancer}

therapy. As aforementioned, telomerase activity can also be inhibited by stabilization of quadruplex conformation of telomeres and, therefore, small molecules that stabilize these quadruplex structures could act as telomerase inhibitors and can be employed as potential therapeutic agents.

Human telomeric sequences are one of the most extensively studied motifs due to their critical role in maintaining chromosomal integrity. Human telomeres consists of highly conserved tandem repeats of guanine rich hexanucleotide d[TTAGGG] ranging from 5-15 kb, while the extreme 3‟ terminus of this telomeric sequence is single stranded and composed of

only about 100-200 bases that folds into a “t-loop” structure[40]_{. The hexanucleotide repeats of}

d[TTAGGG] can fold into an array of quadruplex topologies in vitro under different physiological conditions[5a]_{. Structural information of telomeric quadruplex DNA under in vivo}

conditions is essential from drug design stand point of view. The first structure of the human telomeric DNA sequence, AG3(T2AG3)3 by NMR in Na+ has shown that this sequence folds into

an intramolecular quadruplex termed as an antiparallel basket structure with a mixture of diagonal and lateral TTA loops[41]_{. In the presence of K}+ _{in a crystalline state, an intramolecular}

parallel, propeller-type G-quadruplex conformation has been reported. Propeller-type G- quadruplexes have the loops running diagonally between the G-strands with the G-strands in a parallel arrangement[42]_{. Because the structure in K}+ _{solution is considered to be biologically}

more relevant, due to the high intracellular K+ _{concentration, several attempts have been made}

to elucidate the folding topology of human telomeric quadruplex in K+_{, and several structures}

were found that are inconsistent with the crystal structure. The equilibrium of G-quadruplex species in K+ _{solution can be altered by several additional factors. For example, platinum-based}

cross-linking studies have shown that the basket-type structure coexists with other quadruplexes in both Na+ _{and K}+ _solutions[43]_{. A subsequent} 125_{I-radioprobing study has}

revealed that a chair-type conformation is the major species in K+ _solution[44]_{. Recently,}

sedimentation and fluorescence studies have revealed that the crystal structure of telomeric DNA is unlikely to be the major species in K+ _{solution, and various forms are energetically}

similar [45]_{. A mixture of chair-type and parallel/antiparallel hybrid structures may coexist for}

telomeric DNA in K+ _{solution. Circular dichroism studies of several modified human telomeric}

sequences with bromo-guanine substitutions revealed the formation of highly stable hybrid- type conformations, and were later further confirmed by high resolution NMR studies [46]_.

Recent structural studies by several groups also showed that the human telomeric quadruplex folds into a mixture of “hybrid-type” mixed parallel/antiparallel quadruplex conformations

stabilization, so they cannot be directly compared with the crystal structure. Recently, it was reported that human telomeric DNA forms parallel-stranded intramolecular G-quadruplexes in K+ _{solution under molecular crowding conditions} [48]_{. Moreover, various labs have}

suggested a compact stacking structure for multimers of hybrid-type and parallel-type G- quadruplexes in human telomeric DNA. The “hybrid-type” structures of telomeric quadruplexes under physiological K+ _{conditions maybe the predominant conformation in vivo.}