The investigator proposes to study the molecular structures of two non-sequence specific DNA-binding proteins involved in DNA repair and replication, Thermos aquaticus DNA polymerase and E. coli single strand DNA- binding protein using X-ray crystallography. Free and DNA- bound forms of the proteins will be investigated, as well as site- directed mutants. These studies are expected to provide insight into the mechanisms of DNA polymerization and DNA unwinding. The structure of an active N-terminal deletion of Taq DNA polymerase corresponding to the Klenow fragment of E coli DNA polymerase has been determined to 2.5 Angstroms resolution and will be refined to higher resolution. Additional studies will involve complexes with DNA, deoxynucleotides and dideoxynucleotides in order to provide information on the mechanism of DNA polymerization and will form the basis for attempts to improve the enzyme. The E coli single strand binding protein is essential for DNA replication, DNA repair processes, and in RecA-mediated homologous recombination. A chymotryptic fragment of SSB (SSBc) containing the ssDNA-binding domain has been crystallized and solved at low-resolution. A 2.5 Angstroms native data set, presumably of SSBc, has been collected and efforts are underway to determine a high resolution structure. Selenomethionine has been incorporated into SSB and selenomethionine containing SSBc has been crystallized. The crystals are in the same space group as the native crystals with the same unit cell. Flash- cooling methods have been worked out for these crystals and synchrotron time at the Brookhaven National Laboratory will allow the collection of high resolution multi-wavelength anomalous scattering data. Recently, sizable crystals of a DNA/SSBc complex have been grown.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054033-04
Application #
2910226
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1996-05-01
Project End
2000-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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