UC San Diego

A leading objective in biology is to identify the complete set of activities that each gene performs in vivo. For many years, the telomere biology field has sought define the functional regulatory surface of telomerase, a goal that eluded many researchers due to the lack of structural data for any of the protein subunits. A major goal of this thesis work has been to develop of a rapid genetic approach that can identify amino acids on the surfaces of proteins (even in the absence of structural data) that, when mutated, surgically eliminate single biochemical activities. I used over-expression dominant negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain. This approach is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are therefore able to compete with the endogenous protein (Herskowitz 1987). To test these assumptions, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy (in collaboration with the Wuttke lab), which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts.

Using reverse mutagenesis to target highly conserved charged residues in the ODN assay has been efficient and extremely fruitful. By applying this strategy to all three protein subunits of telomerase, I have been able to define multiple biochemically distinct activities, including identifying two novel surfaces (one in Est3 and one in Est1) required for unknown regulatory functions.

I also supervised several undergraduate students and summer interns to apply the ODN strategy to various essential genes involved in DNA replication and repair, which led to the discovery of EST5. This is the first identification in over twenty years of a gene involved in the telomerase pathway that causes progressive telomere shortening and cellular senescence.