CRYSTALLIZATION AND PRELIMINARY CHARACTERIZATION OF A HYPERTHERMOSTABLE ARCHEAL DNA POLYMERASE. Chen Mao^*, Min Zhou^*, James R. Kiefer^*, Robecca Kucera^**, Maurice Southworth^**, Francine Perler^**, Lorena S. Beese^*. ^*Duke University Medical Center, Durham NC 27710, ^**New England Biolabs, Beverly, MA 01915, USA

A DNA polymerase from a hyperthermophillic marine archeon Thermococcus sp strain 9 N-7 has been cloned, over-expressed, and characterized (Southworth, M.W., Kong , H., Kucera, R.B., Ware, J., Jannasch, H.W., and F.B. Perler, P.N.A.S, 1996 in press). The organism was isolated from a hydrothermal vent chimney scraping on the East Pacific Rise near Galapagos Island at 9 N latitude. The purified polymerase has a half life of 6.7 hours at 95 C and can replicate DNA at temperatures in excess of 85 C in vitro. The enzyme has both DNA polymerase and proofreading 3'-5' exonuclease activities. The polymerase is a 775 amino acid, 89.6 kDa protein, which has significant sequence homology to human DNA Polymerase a (Genbank U47108). Structural elucidation of 9 N-7 DNA polymerase may contribute to our understanding of how DNA is replicated with high fidelity at extremely high temperatures. In addition, it may provide the first structural information about a member of the human Pol a class of DNA polymerases.

Three different crystal forms of this polymerase have been grown. All crystals belong to the orthorhombic Laue group Pmmm, but differ significantly in their cell dimensions. Crystal form I (a=65Å, b= 111 Å, c= 152 Å) diffracts to 3.5 Å resolution and is extremely mosaic (more than 2 degrees). Crystal form II (a= 79 Å, b= 84 Å, c= 134 Å) and crystal form III (a= 97 Å, b= 103 Å, c= 113 Å) both diffract to better than 3 Å resolution and have low mosaicity (less than 0.3 degrees). The estimated solvent content of all three crystal forms is between 50-60% with one molecule per asymmetric unit. Native data sets have been collected from each crystal form. Heavy atom derivative screening is currently in progress.

This work is supported in part by grants from the American Cancer Society (LSB) and the Searle Scholar program (LSB).