The Extrachromosomal Karyoskeleton/Eukaryotic DNA Synthesis and Mutagenesis/Human Neurodegenerative Diseases

Selected Publications
Fisher Lab Page
Medical Scientist Training Program at Stony Brook

The central objective of this laboratory is to understand how extrachromosomal proteinaceous structural elements of the cell nucleus (hereafter-termed karyoskeletal elements) act to regulate nuclear structure and function. Three projects are ongoing. 1) One project entails the identification, biochemical characterization, and functional analysis of major karyoskeletal polypeptides. Along these lines, we are studying interactions between different extrachromosomal karyoskeletal elements, interactions of these elements with interphase chromosomes, the role of karyoskeletal elements in the replication and/or repair of eukaryotic DNA, the molecular events required for disassembly and reassembly of the cell nucleus during mitosis, and the biochemical structure of the nuclear pore complex and its function in the regulation of nucleo-cytoplasmic exchange. Yeast (Saccharomyces cerevisiae) and fruit fly (Drosophila melanogaster) systems permit the genetic analyses essential for studying structural proteins for which in vitro activities are not yet defined. Results obtained to date in Drosophila have been confirmed and/or extended with vertebrate cell types and tissues. This suggests that the Drosophila system is a suitable model for humans. 2) A second line of research deals with the enzymology of eukaryotic DNA polymerases and their specific interactions with protein cofactors. A major focus concerns the polymerase cofactor, proliferating cell nuclear antigen (PCNA), and its role in human mutagenesis. We are also endeavoring to reconstitute various aspects of eukaryotic DNA synthesis in vitro as well as to investigate mechanisms by which DNA polymerases recognize their normal nucleic acid and deoxyribonucleotide substrates, bind them in a precise and highly selective manner, and faithfully catalyze the synthesis of new DNA. Finally, we study recognition, binding and repair of site-specifically damaged DNA. 3) A third project involves a human protein known to cause the neurodegenerative disease, spinocerebellar ataxia (SCA). Nuclear localization of the SCA protein is required for pathology, and pathogenesis presumably involves interaction(s) between the SCA protein and normal nuclear macromolecules. We are currently attempting to identify these putative normal nuclear binding partners of the human SCA protein using a variety of techniques including molecular genetics, co-immunoprecipitation, mass spectroscopy, and chemical crosslinking. Structural studies using X-ray crystallography are also planned.

Selected Publications

Fisher, P.A. (1994). "Enzymologic Mechanism of Replicative DNA Polymerases in Higher Eukaryotes", in Progress in Nucleic Acid Research and Molecular Biology (W. Cohn and K. Moldave, eds.), Vol. 47, Academic Press, NY, pp. 371-397.

Meller, V.H., McConnelL, M., and Fisher, P.A. (1994). An RNase sensitive panicle containing Drosophila melanogaster DNA topoisomerase II. J. Cell Biol. 126: 1331-1340.

McConnell, M., Miller, H., Mozzherin, D.J., Quamina, A., Tan, C-K., Downey, K.M., and Fisher, P.A. (1996). The mammalian DNA polymerase d -prolierating cell nuclear antigen-template-primer complex: Molecular characterization by direct binding. Biochemistry 35: 8268-8274.

Mozzherin, D.J., Shibutani, S., Tan, C.-K., Downey, K.M., and Fisher, P.A. (1997). Proliferating Cell Nuclear Antigen Promotes DNA Synthesis Past Template Lesions by Mammalian DNA Polymerase d . Proc. Natl. Acad. Sci. USA 94: 6126-6131.

Fisher, P.A. and Berrios, M. (1998). "Cell-Free Nuclear Assembly and Disassembly in Drosophilia", in Methods in Cell Biology (M. Berrios, ed.), Academic Press, NY, pp. 397-416.