The genetic damage which accompanies the development and progression of breast cancer has been linked to defects in the DNA synthetic and DNA repair processes of these cells; and these defects correlate with changes in the physical structure and enzymatic activity of several proteins used to carry-out DNA synthesis and repair. The investigator's data indicate that, as compared to normal breast cells, breast cancer cells exhibit a 3-4-fold higher DNA synthetic rate, a 6-8-fold decrease in replication fidelity, and have specific structural alterations in several of the proteins used to carry-out DNA replication. His goal, therefore, is to establish a clear link between the differences in the DNA synthetic activity and replication fidelity of normal and malignant breast cells and the structural changes specific replication essential proteins undergo during transformation. To accomplish this, he will initially focus on defining the kinetic, biophysical, and detailed structural properties of the DNA polymerases and poly(ADP-ribose) polymerase (PARP) by determining: 1) differences in the kinetic properties of these enzymes between the normal and malignant breast cells looking specifically at the Km and Vmax for substrate utilization, substrate specificity, and the proof-reading activity of the intrinsic 3-5' exonuclease of DNA polymerases delta and epsilon; as well as the fidelity with which all of the DNA polymerases carry-out DNA synthesis; 2) differences in the biophysical characteristics of normal and malignant breast cell DNA polymerases alpha, delta, epsilon and PARP looking specifically at the molecular weight, Stokes radii, sedimentation coefficient, isoelectric point, and 2D-PAGE profiles of these enzymes; and 3) differences in the 2D-PAGE tryptic peptide maps of the normal and malignant breast cell polymerases looking specifically at the role of phosphorylation, poly(ADP-ribosylation), and primary amino acid sequence in mediating these differences.
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