Telomeric DNA

Neidle S, Parkinson GN (2003) The structure of telomeric DNA. Curr Opin Struct Biol 13(3) 275-283... 

Parkinson GN, Lee MP, Neidle S (2002) Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417(6891) 876-880... 

Ishikawa F, Matunis MJ, Dreyfuss G, Cech TR (1993) Nuclear proteins diat bind die pre-mRNA 3 splice site sequence r(UUAG/G) and die human telomeric DNA sequence d(TTAGGG)n. Mol Cell... 

Recently, a human sperm H2B was identified that is part of protein complex that specifically binds the telomere DNA repeat [123]. This telomere-binding complex is extracted by conditions (a solution containing 0.5% Triton X-100 and 100 mM NaCl) that do not extract nucleosomal H2B, suggesting that this H2B is not extracted from a typical nucleosomal structure. The structure of this sperm H2B has not been determined. 

Konig, R, Giraldo, R., Chapman, L. and Rhodes, D. (1996). The crystal structure of the DNA-binding domain of yeast RAPl in complex with telomeric DNA. Cell 85, 125-136. 

The thing that has attracted most attention to telomeric DNA is the unusual structure of the G-rich strand that was signaled by the first NMR studies.259 Subsequent investigation260 261 revealed the presence of G quartets (Fig. 5-8) which are apparently stacked in folding patterns such as the following 255 262 268... 

As mentioned in Section B, 1, human centromeres are rich in the repetitive a-satellite DNA. By joining a-satellite DNA-containing fragments of the X-chro-mosome to cloned telomeric DNA, human minichromosomes have been created.488 These have been developed into human artificial chromosomes,489 which may be practical vehicles for gene transfer in human therapy. 

Kang, C., X. Zhang, R. Ratliff, R. Moyzis, and A. Rich, Crystal structure of four-stranded Oxytricha telomeric DNA. Nature 356 126-131, 1992. 

Since eukaryotic chromosomes are linear, the ends of these chromosomes require a special solution to ensure complete replication. This can be seen in figure 26.26. At the very end of a linear duplex a primer is necessary to initiate DNA replication. After RNA primer removal there is bound to be a gap at the 5 end of the newly synthesized DNA chains. Since DNA synthesis always requires a primer the usual way of filling this gap is not going to solve the problem. This dilemma is overcome by a special structure at the ends (telomeres) of eukaryotic chromosomes and a special type of reverse transcriptase (telomerase) that synthesizes telomeric DNA. In many eukaryotes the telomeres contain short sequences (frequently hexamers) that are tan-demly repeated many times. Telomerase contains an RNA that binds to the 3 ends and also serves as a template for the extension of these ends. Prior to replication, the 3 ends of the chromosome are extended with additional tandemly repeated hexamers. The 3 ends are extended sufficiently so that there is room to accommodate an RNA primer. In this way there is no net loss of DNA from the 5 ends as a result of replication. After replication the 3 end is somewhat... 

Williamson, J. R., Raghuraman, M. K., Cecil, T. R., Mono valent cation induced structure of telomeric DNA - the G-quartet model. Ceil 1989, 59, 871-880. 

Fig. 3. Replication of telomeric DNA. Telomerase has a bound RNA molecule that is used as template to direct DNA synthesis and hence extension of the ends of chromosomal DNA.

The interactions of the G-quadruplex of human telomere DNA with these newly designed molecules have been examined via CD spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative and G-quadruplex or duplex DNA has been investigated by... 

The end of a linear chromosome is called a telomere. Telomeres require a special mechanism, because the ends of a linear chromosome can t be replicated by the standard DNA polymerases. Replication requires both a template and a primer at whose 3 end synthesis begins. The primer can t be copied by the polymerase it primes. What copies the DNA complementary to the primer In a circular chromosome, the primer site is to the 3 direction of another polymerase, but in a linear chromosome, no place exists for that polymerase to bind. As a result, unless a special mechanism for copying the ends of chromosomes is used, there will be a progressive loss of information from the end of the linear chromosome. Two characteristics about telomeres help avoid this situation. First, they consist of a short sequence—for example, AGGGTT—repeated many times at the end of each chromosome. Telomeres, therefore, are part of the highly repetitive DNA complement of a eukaryotic cell. Secondly, a specific enzyme, telomerase, carries out the synthesis of this reiterated DNA. Telomerase contains a small RNA subunit that provides the template for the sequence of the telomeric DNA. Eukaryotic somatic cells have a lifespan of only about 50 doublings, unless they are cancerous. One theory holds that a lack of telomerase in cells outside the germ line causes this limitation. 

Much like the search for protein-binding compounds requires consideration of tertiary structure in addition to peptide sequence, thinking about selective nucleic acid binding solely in the context of primary sequence is often an oversimplification. In the context of DNA recognition, a particularly important example of this is the case of G-quadruplexes. As their name implies, these structures consist of stacked tetrads of guanosine bases, typically ordered around a monovalent cation. One representative structure, a quadruplex derived from human telomeric DNA, is shown in Fig. 5 many variants of this motif formed by either parallel or antiparallel DNA strands have been observed. 

Fig. 5 Top view of a representative G-quadruplex X-ray crystal structure (human telomeric DNA PDB ID 1KF1 [7]. Guanosine bases forming tetrads are at the center of the structure in this view...

Telomerase is a ribonucleoprotein complex that exists in eukaryotic cells for the apparently sole purpose of synthesizing telomeric DNA, which consists of tandemly repeated sequences that contain clusters of G-residues and forms the ends of chromosomes. Telomerase comprises two essential core components, a protein subunit that has reverse transcriptase (RT) activity and an RNA sequence (hTR) that contains clusters of C-residues and serves as the template substrate for the RT (6). The G-rich DNA and C-rich RNA anneal to form a partial duplex with DNA as the primer. RT-mediated polymerization of dGTP and other complementary triphosphate substrates produces a DNA terminus that has been extended by around six nucleotides. The new end can become a substrate for either another round of telomerase-mediated elongation or primase/polymerase-mediated lagging-strand synthesis. 

The first clue to how this problem is resolved came from sequence analyses of the ends of chromosomes, which are called telomeres (from the Greek telos, "an end"). Telomeric DNA contains hundreds of tandem repeats of a... 

Figure 27.36. Telomere Formation. Mechanism of synthesis of the G-rich strand of telomeric DNA. The RNA template of telomerase is shown in blue and the nucleotides added to the G-rich strand of the primer are shown in red. [After E. H. Blackburn. Nature 350(1991) 569.]...

DNA showed that there might be a biological consequence to oxidation of telomeric DNA. Telomeric DNA contains GGG sequences, and telomeres adopt a quadruplex structure. Oxidation of the 5 -G resulted in a quadruplex structure, but oxidation in the middle of the GGG triplex led to multiple structures. Telomerase activity was significantly reduced when 8-oxo-dG was at the 5 -end of the triplet, but not in the middle where multiple structures were formed. 

A number of oligonucleotide hairpin, quadruplexes and other higher ordered tertiary structures have also been reported the solution structure of the transcriptional antiterminator LicT from Bacillus subtilis bound to a 29 bp ribonucleic antiterminator RNA hairpin " a crystal structure of a kissing complex of the HIV-1 RNA dimerisation initiation site " the crystal structure of the Za high affinity-binding domain of the RNA editing enzyme ADARl bound to left-handed Z-DNA " the crystal structure of parallel quadruplexes from human telomeric DNA. " ... 

Human telomerase is a structurally complex ribonucleoprotein that is responsible for the maintenance of telomeric DNA at the ends of chromosomes. Telomerase acts to synthesize and add a simple six-base motif (of TTAGGG in the human case) to the ends of the chromosomes, resulting in stable telomere length that would otherwise be gradually eroded after each cell replication. Active telomerase has been detected in a majority of human cancer, embryonic, and germline cells but not in normal somatic cells, with the exception of some stem cells, such as those involved in tissue renewal. 

Last, the hTERT reverse transcriptase inhibitors do not effect their activity by specifically and persistently binding to hTERT rather, they act as competitors for the substrate deoxyribonucleotides used by reverse transcriptases, such as hTERT, to construct DNA chains (or more specifically for hTERT, to construct telomeric DNA extensions). Small nucleoside analogues can act as reverse transcriptase inhibitors, although only some of these compounds, such as 6-thio-2 -deoxyguanosine 5 -triphosphatc (TDG-TP), are selective against the hTERT reverse transcriptase (72). TDG-TP is effective at low micromolar concentrations (72) and stops telomeric DNA extension after incorporation into the DNA (73). 

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