David Zappulla Assistant Professor Department of Biology CMDB Graduate Program Faculty B.A. Middlebury College Ph.D. Stony Brook University Postdoctoral University of Colorado, Boulder

[Research Interests] [Representative Publications] [Lab Members]

RESEARCH INTERESTS

The telomerase RNA-protein complex, chromosomes and aging

Eukaryotic nuclear chromosomes are linear and therefore each have two ends, called telomeres. Discovered in 1984, the enzyme telomerase synthesizes telomeric DNA by reverse transcription of an intrinsic RNA subunit and is required for completion of chromosome replication and the stability of the genome in many eukaryotes including vertebrates, yeasts, and ciliated protozoa. Telomerase plays a central role in permitting unlimited human cancer cell proliferation and also plays a role in the aging process. A major aim of our research is to understand the functions and coordination of the telomerase ribonucleoprotein (RNP) enzyme complex subunits as well as how telomerase activity at the ends of chromosomes is regulated.

Our previous research has shown that the budding yeast Saccharomyces telomerase RNA, TLC1, is evolving very rapidly; perhaps even more so than in other evolutionary branches, and extraordinarily faster than other essential ncRNAs, even in yeast. Why is this the case? Some insight into this question has also come from deletion analysis that has shown that two-thirds of S. cerevisiae telomerase RNA is dispensable for function in vivo and that protein-binding sites in the RNA (including essential ones) can even be repositioned to very unnatural locations in TLC1 with retention of function. The sum of the evidence has lead us to conclude that the large yeast telomerase RNA functions as a flexible scaffold or tether for protein subunits, thus defining a new class of RNP complexes. Interestingly, the large size allows greater fitness of yeast as demonstrated by competitive culture experiments between wild-type cells and those expressing a miniaturized telomerase RNA called “Mini-T,” thus suggesting why it has evolved. We continue to investigate the attributes of flexible scaffolding of protein subunits by RNA in this RNP as well as other aspects of telomerase architecture.

The compact Mini-T RNA has afforded us the ability to reconstitute yeast telomerase activity in vitro, which has never been possible using wild-type 1157-nt TLC1 (probably because the big RNA misfolds in vitro, being trapped in “alternate conformer hell”). Using this reconstituted yeast mini-telomerase activity assay in parallel with purified wild-type telomerase from yeast extracts (as well as experiments in vivo), we are investigating yeast telomerase activity. We have shown that the yeast core enzyme, comprised of the RNA and the telomerase reverse transcriptase subunit (TERT; Est2 in yeast), is intrinsically nonprocessive. This contrasts with the highly processive human enzyme. However, yeast telomerase does readily add multiple telomeric repeats in vivo to a single telomere. Thus, we aim to understand what is required for significant repeat addition by yeast telomerase. Our main approach is to add purified protein factors into the reconstituted system to test for functions of individual components. However, forward genetics in yeast is another powerful tool that we employ to identify important components involved in this process.

Another interest is in the processes of aging and senescence. What governs the progression of these pathways and can it be modulated? What is the role of telomerase in these phenomena? Is the aging process intrinsic to cellular life, or has it evolved? Such questions are of great interest and devising ways to test them in yeast and other systems is a goal for future endeavors.

REPRESENTATIVE PUBLICATIONS

Zappulla, D.C.*1, Roberts, J.N.*, Goodrich, K.J., Cech, T.R. and Wuttke, D.S. 1 (1 co-corresponding author, *co-first author) 2009. Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Research. 37:354–367.

Box, J.A., Bunch, J.T., Zappulla, D.C., Glynn, E.F., and Baumann, P. 2008.  A flexible template boundary element in the RNA subunit of fission yeast telomerase. Journal of Biological Chemistry. 283:24224–33.

Zappulla, D.C. and Cech, T.R. 2006. RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harbor Symposium on Quantitative Biology. 71:217–224.

Zappulla, D.C., Maharaj, A.M., Connelly, J.J., Jockusch, R., and Sternglanz, R. 2006. Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs. BMC Molecular Biology. 4:40–52.

Zappulla, D.C., Goodrich, K., and Cech, T.R. 2005. A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nature Structural and Molecular Biology. 12:1072–1077.

Zappulla, D.C. and Cech, T.R. 2004. Yeast telomerase RNA: a flexible scaffold for protein subunits. Proceedings of the National Academy of Sciences. 101:10024–10029.

Andrulis, E.D., Zappulla, D.C., Alexieva-Botcheva, K., Evangelista, C. and Sternglanz, R. 2004. One-hybrid screens at the Saccharomyces cerevisiae HMR locus identify novel transcriptional silencing factors. Genetics. 166:631–635.

Zappulla, D.C., Sternglanz, R., and Leatherwood, J. 2002. Control of DNA replication timing by a transcriptional silencer. Current Biology 12:869–875.

Andrulis, E.D.*, Zappulla, D.C.*, Ansari, A.*, Perrod, S., Laiosa, C.V., Gartenberg, M.R., and Sternglanz, R. (* equal contribution). 2002. Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning. Molecular and Cellular Biology. 22: 8292–8301.

Andrulis, E.D., Neiman, A.M., Zappulla, D.C., and Sternglanz, R. 1998. Perinuclear localization of chromatin facilitates transcriptional silencing. Nature. 394:592–595.

Tufarelli, C., Fujiwara, Y., Zappulla, D.C., and Neufeld, E.J. 1998. Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene. Developmental Biology. 200: 69–81.

Yandava, C.N., Zappulla, D.C., Korf, B.R., Neufeld, E.J. 1996. ARMS test for diagnosis of factor VLeiden mutation, a common cause of inherited thrombotic tendency. Journal of Clinical Laboratory Analysis. 10:414-7.

Lab Members

PhD Student:
Kevin Lebo

Postdoctoral Fellow:
Melissa A. Mefford

Research Technologist:
Qundeel Rafiq