STRUCTURES OF NASCENT DUPLEX DNA BOUND TO A THERMOSTABLE DNA POLYMERASE AT 1.9 Å RESOLUTION. Lorena S. Beese, Chen Mao, James R. Kiefer, Stephen B. Long, and Jeff Braman*. Dept. of Biochemistry, Duke University Medical Center. *Stratagene Inc., La Jolla, CA 92037

Co-crystal structures of a thermostable 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 Å 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. We 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 primarily B-form conformation, however the minor groove widens as the DNA enters the polymerase cleft. No bend in the DNA is necessary 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. cold 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 it-factor of the refined structure is 19.6% between 8 A and 2.2 Å resolution (Rfree=25%) with 0.010 A rms deviation in bond lengths and 1.5 deg. rms 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 Å resolution to an R factor of 19.9% (Rfree=25%) with 0.009 rms deviation in bond lengths and 1.5 deg. rms deviation in bond angles.

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