Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes that are protected and maintained by specialized proteins including the reverse transcriptase telomerase. Telomere defects have been linked with both aging and transformation. A critical activity at telomeres is the proper capping, and processing, of the conserved single-stranded overhang. In humans the overhang of repeats of d(TTAGGG) can be up to 300 bases long. This 3'end can invade the double stranded portion of the telomere, forming protective loops. Disruption of the cell's ability to discriminate the end of the telomere from damaged DNA is catastrophic and leads to severe genomic instability. Access of telomerase to the overhang is strongly controlled and telomere maintenance is a strong regulator of the proliferative potential of the cell. Disruption of telomere maintenance is a potential means for therapeutic intervention. This proposal is based on the hypothesis that vertebrate telomere repeat DMAs form quadruplex structures, favoring those with external loops that are also known as propeller structures, in the presence of the high concentrations of potassium and low concentrations of sodium that are present in nuclei. The formation of external loop structures poses fewer topological problems than the formation of other quadruplex structural types. The ligands designed and screened to target telomere DMAs should be aimed at external loop structures. The protection that the external loop structures can provide to nuclease and chemical attack will be determined.
Specific Aim 1. Methods for the determination of quadruplex structure type will be validated using DMAs whose structures have been determined by NMR. The methods to be validated include circular dichroism and hydroxyl radical cleavage. The validated methods will be used to determine the structures of vertebrate telomere DNAs. The structural results will be important in determining which types of quadruplex DNA can be present at the telomere and how the length of the vtrDNA that is uncapped may alter the structure and maintenance of the human telomere.
Specific Aim 2. The validated methods will be applied to investigating the effects of porphyrin binding on the structure and reactivity of vertebrate telomere DNAs. These studies include DNAs with more than one quadruplex unit since the effects of porphyrins may be to disrupt the interactions between quadruplex units.
Specific Aim 3. The effects of di- and multivalent cations on the quadruplex structures and the equilibria between the quadruplex structures of vertebrate telomere repeat DNAs will be determined using the validated methods.
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