Project Title: 15. Molecular and Cellular Radiobiology . The Chemistry and Functjon of DNA Polymerases Relationship to Other Projects: RX-03-02- (a) (Cont'd.) the virus-like particle originally derived from a spontaneous rat mammary tumor. At BNL a unique opportunity exists to study, on a molecular level, induced tumors produced in another study (Shellabarger RX-03-Ol-a). 16. radiation Technical Progress in FY 1973: From chick embryo skeletal muscle (legs), the hybrid template-directed DNA polymerase has been purified approximately 100 fold by a three-step procedure involving ammonium sulfate fractionation, DEAE cellulose chromat- ography,and phosphocellulose chromatography. During the course of this purification other DNA polymerases which prefer as template “activated" or denatured DNA are removed, A ' Although it was believed a year ago that two different enzymes-- one soluble, the other membrane-bound--occur in the chick embryo, this opinion is no longer tenable. During the course of the above purification the enzyme in the soluble fraction and the enzyme subsequently extracted by Triton X-100 behave more and more the same. There is now no reason to believe that the two preparations differ in any important way. When first extracted from the tissue the enzyme appears to have a very high molecular weight (perhaps 2 x 106, determined by gel filtration). With purification the apparent molecular weight becomes smaller, passing ‘ through one or more intermediates, so that after the phosphocellulose step the MW is about 30,000. The nature of this transition and the exact size of the purified enzyme is still under study. . Methods for purification of nuclei of various chick embryo tissues have been worked out and preliminary studies on the subcellular distribution of hybrid-dependent DNA polymerase activity have been made. Considerable variation has been observed from tissue to tissue, For example, in liver, 58% of the total activity was found in the cytoplasm whereas in heart tissue more than 90% was in the cytoplasm. The significance and generality of this observation is under continuing study. As another part of the effort to understand the biological significance of hybrid template-dependent DNA polymerases, fluctuations in enzyme level have; been studied in synchronized HeLa cells. Again, enzyme activity was “Both in the nucleus and cytoplasm. Variation in total cellular enzyme was small compared to variation in the rate of DNA synthesis, During Mod of rapid DNA synthesis (S phase), enzyme specific activity (cpm incorporated/mg protein) was approximately 50% higher than before G) or after Go DNA synthesis. During S phase, while the rate of DNA synthesis However, change in enzyme specific activity. significant no was there doubled, a larger proportion of the total enzyme activity was found in the nuclear fraction during S phase than in G, or G9. (See Continuation Sheet) 1119280 RX-220