STRUCTURE & FUNCTION OF BASE MODIFICATIONS IN SUBTELOMERIC & TELOMERIC DNA

Washington University, Saint Louis MO

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Normal human cells have a limited capacity to proliferate, a process termed replicative aging. Increasing evidence has implicated telomeres, the structures that cap the ends of the chromosomes, as the molecular clock that counts the number of times the cell has divided. The mechanism of lagging-strand DNA synthesis prevents DNA polymerase from replicating the DNA all the way to the 5''''end of a linear chromosome, leaving a 3''''overhang and causing the chromosomes to shorten every time a cell divides. Human telomeres are composed of many kilobases of the repetitive sequence TTAGGG that, together with telomere-binding proteins, prevent the cell from recognizing the end of the chromosome as a DNA break needing repair. Cellular senescence may occur when some of the telomeres have shortened sufficiently to induce a DNA damage signal. Cancer cells escape the proliferative limits of replicative aging by up-regulating the expression of telomerase, an enzyme capable of adding telomere repeats to the ends of the chreomsomes and maintaining their length. Using methods for identifying the presence of modified nucleotides in subtelomeric DNA, for purifying telomeres (based on the presence of the 3''''G-rich overhang) that yields a greater than 1000-fold enrichment in a single step, for determining the size of the overhangs, and for measuring telomere sizes in interphase nuclei. Chromatographic separation for the telomeric nucleobases has been developed. It is a HILIC based separation appropriate for these very hydrophilic compounds and it also affords high mass spectrometric sensitivity. The method has been adapted to be used on nano-LC columns and nano electro spray ionization to increase the sensitivity