1) Background: Exposure of biologically active materials to ionizing radiation at very low temperature leads to damage to macromolecules. Biochemical functions are lost at a rate directly dependent on the molecular mass of the active structures. This is the basis for radiation target analysis which is used to determine the size of the functional unit of enzymes, receptors, transporters and others that carry out important biological activities. These studies revealed new and unexpected aspects of biomedically important systems including basic differences between proteins and nucleic acid in their responses to ionizing radiation. 2) Objective of present studies: a: Fundamental studies of the actions of ionizing radiation on macromolecules continue in order to define the exact nature of the damage in different species of molecules. Analyses of these effects by radiation target theory establishes a radiation-sensitive mass associated with measured biological activities. This reveals a fresh perspective in the structure-function relationship in these macromolecules. b: Application of the radiation technique to enzymes, binding sites, and transporters to determine the size of their active structures, which often is less than the mass of the entire complex. 3) Results during the past year: ? a. Radiation target analyses of DNA have been completed. One study involved the primer and template DNAs associated with reverse transcriptase. Radiation inactivation of the protein portion of this enzyme had previously been reported. Two small singe-stranded DNA molecules of 5 kDa and 10 kDa base-pair with each other in the function of this enzyme. The inactivation of each single-strand has been followed individually and both strands revealed a radiation target size of 15 kDa, indicating that there is radiation energy transfer between the two polynucleotides. The loss of biological activity was also found to yield the same target mass. Another project involved supercoiled DNA; cleavage of the backbone of one strand leads to an uncoiling of the molecule to an 'open circle' form, a circular molecule with a broken bond in the backbone of one strand. A second break in the opposite strand, close to the original break in the first strand destroys the circular structure and the molecule assumes a linear conformation. The three forms (supercoiled, open circle and linear form) can be resolved by gel electrophoresis. Irradiated supercoiled DNA disappears as a simple exponential function of radiation dose; the quantity of open circle molecules increases at low radiation exposures as they are formed from damaged supercoiled molecules. After greater exposures, the amount of open circle molecules slowly decreases exponentially with radiation dose. The formation and destruction of the linear form as well as biological function (transfection) have also been determined in the same samples. ? b: The restriction of radiation damage to a single position RNA was ascribed to the presence of sugar rings in the polynucleotide backbone. ? c: Radiation target analysis of polysialyltransferase has been completed. The enzyme synthesizes a high molecular weight polysaccharide forming a capsule in gram negative bacteria, permitting the organism to avoid detection by the host immune system. A polysialic acid gene cluster is comprised of several genes proposed to encode a biosynthetic complex; the overall polmerization consists of two reactions: (a) the transfer of sialic acid to a primer, and (b) elongation of the initiated chain. ? d: Certain endothelial cells synthesize and excrete a lipase which displays both phospholipase and triglyceridase activities. In vivo the enzyme is partially cleaved enzymatically. Preparations in which the cleavage is inhibited show a larger target size. The enzyme might be functioning as a dimer - as in lipoprotein lipase and hepatic lipase.? 4) Conclusions and significance. Radiation target analysis can be applied to DNA. In the radiation studies of supercoiled DNA, results suggest surprises about the nature of radiation energy transfer between and along individual DNA strands. The radiation sensitivity of primer and template DNA strands in reverse transcriptase clearly show transfer of radiation-deposited energy between the two chains. In studies of the large DNA plasmids, the target size for the disappearance of the open circle DNA form indicates something about the distance between the original break in the opposing strand and the new break in the other strand. Either the two breaks do not have to be 'within a few bases' as previously believed, or else some of the radiation-deposited energy can be transferred along a stretch of several hundred bases. This latter possibility is in contrast with previous observations with single-stranded RNA where the transfer distance was at most 3-5 bases.
