DNA POLYMERASE FIDELITY: A STRUCTURAL ANALYSIS. M.M. Blair, N. Mashhoon, C.R.A. Muchmore, M.F. Goodman, and W.F. Anderson, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611 and Department of Molecular Biology, USC, Los Angeles, CA 90089
E.coli DNA Polymerase II (pol II), an 89.9 kD, [[alpha]]-like DNA polymerase, possesses both polymerase and 3'-5' exonuclease activities on a single polypeptide chain. Pol II is induced in response to DNA damage as part of the SOS regulon in E.coli and is required for synthesis past abasic lesions in the absence of heat shock proteins. Recent in vivo studies suggest that replacing wild type with exonuclease deficient pol II leads to increased adaptive mutation frequency indicating an important role for pol II in replication fidelity in the cell. To better understand the mechanism of fidelity, we are using x-ray crystallography to study mutants of pol II and their complexes with DNA.
Mutants of pol II, D155A/E157A (exo-) and L423M, were constructed. These mutations are in highly conserved regions of Pol II involved in nucleotide/metal binding. Pol II exo- has wild type levels of polymerase activity but lacks exonuclease proofreading activity. The L423M mutant has wild type levels of polymerase and exonuclease activities but partitions preferentially towards polymerization in the presence of a mispaired primer terminus suggesting an alteration in switching between the polymerase and exonuclease sites.
Both mutants have been crystallized by vapor diffusion methods. Like Pol II, Pol II exo- crystallizes in the P21212 spacegroup and both room temperature and low temperature data sets have been collected. Initial phasing of pol II exo- was by low resolution, modified MIR phases from room temperature pol II data. Further work is underway to improve phases via derivative searches, model building, and density modification.
Pol II wild type and exo- mutant have also been co-crystallized with synthetic double-stranded DNA oligonucleotides. Further work is in progress.
This work is supported by NIH Grant GM15075, GM48569 and ES05355.