Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant
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Telomeres, the ends of linear chromosomes, are composed of simple tandem repeats which are usually G·C rich. Telomeres are essential for chromosome stability, organizing the nuclear architecture and ensuring complete replication of the chromosomal terminus. To understand how telomeres carry out these fundamental cellular roles, one must understand the structural and dynamic properties of telomeric repeat sequences. Structural and genetic approaches were taken to learn more about telomeric nucleic acids;The structural portion of my research concerned an unusual DNA structure formed by the C-rich strand of telomeric DNA. Telomeric C-strand sequences form non-Watson-Crick structures in supercoiled plasmids at low pH. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoresis showed that telomeric C-strand oligonucleotides form stable structures at low pH. H1[superscript]'-H1[superscript]' nuclear Overhauser effects indicated that these structures were four-stranded. In addition, these four-stranded C-structures were shown to mediate recognition and binding of identical nucleic acid sequences. Thus, a novel nucleic acid dimerization motif was discovered;The genetic portion of my research concerned analysis of a Tetrahymena mutant with short telomeres. This mutant was heterozygous for a telomerase RNA mutation. Telomerase is a ribonucleoprotein that uses its RNA component as a template for addition of telomeric repeats to chromosome termini. Therefore, telomerase is involved in telomere length regulation, a process that has been implicated in both aging and cancer. The mutant telomerase RNA gene caused telomere shortening when introduced into wildtype cells, and thus identifies a functionally important domain of the telomerase RNA. Although mutant telomerase activity was indistinguishable from wildtype activity in vitro, cells expressing high levels of the mutant telomerase RNA exhibited lethal phenotypes that were due to the presence of very short telomeres.