Human chromosomes end in repetitive DNA sequences bound by the shelterin complex. During DNA replication chromosomes shorten because of the failure to copy the very end of the chromosome. If the chromosomes shorten too much cells stop dividing. This mechanism limits the number of divisions human cells can undergo and prevents them from turning into cancer cells. In stem cells and germ cells, which are cell types that have to divide continuously throughout a humans life, telomerase, an RNA containing reverse transcriptase, adds telomeric repeats back to the chromosome end to maintain telomere length, allowing these cells to keep proliferating. Unfortunately, 90% of cancers take advantage of telomerase to facilitate their continuous growth, making telomerase an attractive target for developing new treatment strategies for a large number of malignancies. Our goal is to quantitatively define the molecular mechanisms that underly telomerase recruitment to telomeres and telomere elongation. Understanding the molecular details of telomerase mediated telomere maintenance could lead to new approaches that target telomerase specifically in cancers cells.
To achieve this goal we have developed genome editing strategies, that allow us to modify any gene in human cells. We use this approach to append fluorescent tags to proteins of interest, at their endogenous genomic loci, to assure that their expression and its regulation is as similar as possible to the proteins untagged counterpart. These genome edited cell lines can then by subjected to live cell single molecule imaging, a powerful technique that can detect individual proteins in human cells to analyze their behavior in real-time. Using this method we make precise measurements of the location, rate of movement, and residence time of the protein of interest, for example telomerase. In combination with biochemical data, our goal is to use this information to build a quantitative model of telomerase mediated telomere maintenance. We can then compare normal human cells with cancer cells to determine whether there is any cancer specific aspects to this important process, which could potentially be targeted to interfere with telomerase in cancer cells.