Hematoporphyrin derivative (HpD) and light have been used as an effective antitumor therapy. The drug causes chromosomal DNA damage in mammalian cells at low doses, and inhibits DNA repair at higher doses. We have recently shown that purified calf DNA polymerase delta II is inhibited at therapeutic HpD concentrations (approx. 10-6 M), which do not affect the other major cellular DNA polymerases alpha and delta I. The delta forms I and II are the only mammalian DNA polymerases that contain a 3' to 5' exonuclease, an activity thought to be necessary for accurate DNA replication and repair. We now propose to identify the enzymes responsible for repair of HpD damage to chromosomal DNA, and to determine whether HpD induced inhibition of DNA polymerase delta II is responsible for inhibition of this repair. We will expose Chinese hamster ovary cells to low doses of HpD and light, and then measure DNA repair in the presence of specific inhibitors of DNA polymerases alpha, delta I, delta II, beta and gamma, and topoisomerases I and II. Prevention of repair processes by one of these specific inhibitors will indicate a requirement for the inhibited enzyme. Extracts of cells exposed to higher doses of HpD and light will be assayed for the activities of each of these DNA replication and repair enzymes so that the inhibitory effects of HpD can be evaluated. The chemical nature of HpD damage to DNA before and after processing by cellular DNA repair enzymes will be determined. DNA damaged by HpD will be evaluated for its ability to serve as a template for synthesis by calf nuclear polymerases, alpha, delta I and II, and beta, each purified to near homogeneity in our laboratory. The chemical mechanism by which HpD inactivates DNA polymerase delta II will also be investigated. Results should clarify the mechanism by which HpD inhibits DNA repair, and should also define the biological role of DNA polymerase delta II.
