Co-crystal structures of a thennostable DNA polymerase from a newly identified strain of Bacillus spp with two different DNA primer-templates bound in the active site were determined at 1.9 A resolution. This is the first crystal structure of a DNA polymerase in the Pol I family with duplex DNA bound at the polymerase active site. vVe were able to deduce the direction of DNA synthesis by comparing the structures of a primer template complex and an extended primer template complex in which an additional nucleotide was added by the polymerase in the crystal. The -3' hydroxyl of the primer strand interacts with a highly conserved, catalytically important aspartate. The duplex DNA adopts a primar·ily B-form conformation, however the minor groove widens as the DNA enters the polymerase cleft. No bend in the DNA is necessar-y to reach the polymerase active site. A network of hydrogen bonds is made between the sugar-phosphate backbone of the DNA base pairs and highly conserved residues of the protein. The direction of DNA synthesis is consistent with the model proposed based on the editing complex of the E. coli Klenow fragment. These observations unambiguously resolve a recent controversy about the direction of DNA synthesis in the Pol I class of DNA polymerases.The crystal structure of the apo DNA polymerase was determined by the method of multiple isomorphous replacement including the anomalous scattering data from two heavy atom derivatives. The R-factor of the refined stmcture is 19.6% between 8 A and 2.2 A resolution (Rfree=25%) with 0.010 Arms deviation in bond lengths and 1.5 a m1s deviation in bond angles. The structures of the DNA complexes were determined by molecular replacement using the apo structure as a starting model. These structures are refined at 1.9 A resolution to an R factor of 19.9% (Rfree=25%) with 0.009 rms deviation in bond lengths and 1.5 a rms deviation in bond a11gles.This work is supported in part by grants from the American Cancer Society (LSB) and the Searle Scholar program (LSB).MS04.05.05 STRUCTURAL STUDIES OF TYPE I DNA TOPOISOlVIERASES Alfonso Mondragon, Christopher Lima, Neal Lue, Alexandra Patera, and Amit Sharma. Depar·tment of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA DNA topoisomerases are proteins responsible for controlling and maintaining the topological state of DNA in the cell. They have been found in all cell types of both eukal}'otes and prokaryotes and additionally in some viruses. They ar·e involved in DNA replication, transcription, and genetic recombination. All topoisomerases work by fomring a tra11sient break in DNA through a phosphotyrosine bond. Type I topoisomerases break one DNA strand at a time and then pass another strand through the transient break. No external energy source is required for this reaction as the bond energy is conserved.We have identified and cr-ystallized a 67 kDa domain of Escherichia coli DNA topoisomerase I containing the catalytic tyrosine that is capable of cleaving single st...

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