Fine-Tuning Density Modification For DNA Polymerase II From E. Coli. C.R.A. Muchmore, M.M. Blair, L. Shuvalova, N. Mashhoon, M.F. Goodman, W.F. Anderson, Northwestern University Medical School, Department of Molecular Pharmacology and Biological Chemistry, Chicago, Illinois and University of Southern California, Department of Molecular Biology, Los Angeles, California

This x-ray structure solution was hindered by the most common problems in ab initio phase determination by MIR methods: Low resolution starting phases, and only 43% solvent content without any non-crystallographic symmetry. With phases from one Hg derivative to 3.5Å and two Pt derivatives to 4.5Å and 4.0 Å resolution, the location of helices in the protein density was obvious in a 6.0 Å map. Careful adjustment of parameters in phase refinement and extension produced an improved 3.2Å resolution map, in which beta structure is visible and the direction of helices is recognizable. This map was used with the program ESSENS to locate secondary structural elements. The resulting ESSENS score maps were back-transformed as if they were electron density maps and the resultant phases combined with the MIR phase probability distribution with SIGMAA (CCP4). Phase refinement and extension using these combined phases to calculate the starting map resulted in improved map quality.

Details of this phase improvement procedure, map quality at the individual steps and the current model for DNA Polymerase II from E. coli. will be presented.

Polymerase II from E. coli is a member of the group B (a-like) DNA polymerases as indicated by conserved sequence motifs. It contains both polymerase and 3'->5' exonuclease activity on a single polypeptide chain of 781 amino acid residues and is induced by DNA damage. The [beta][gamma] accessory protein complex of the polymerase III holoenzyme increases polymerase II processivity, raising questions about the nature of this interaction. Information about structure and function derived from the atomic structure of E. coli polymerase II may be applicable to other homologous group B polymerases like mammalian cellular DNA polymerase a, eucaryotic viral polymerases and bacteriophage T4 polymerase.

This work is supported by NIH GM48569, ES05355 and GM15075 .