This project has two major objectives. One is to examine the structure of mammalian DNA polymerase alpha (pol alpha) and its putative function in nuclear DNA replication, using Chinese Hamster Ovary (CHO) cells as the model system. The second is to develop further, through synthesis and systematic evaluation of structure-activity relationships, the pol alpha-specific anilinouracil inhibitors recently characterized in our laboratory. The development of pol alpha-specific anilinouracil inhibitors will include: (1) the examination of the spectrum of mammalian cells and mammalian pol alphas which are sensitive to the drug prototype and (2) the screening of structurally modified forms of the prototype to secure derivatives with increased potency and utility in vivo and in vitro. The examination of the cellular function of pol alpha will attempt: (1) to determine if pol alpha is a replication-specific enzyme, and (2) to sort out the relationships of the distinct species of alpha-like polymerases present in cell extracts. The approach will rely on the selection of mutant cells resistant to the pol alpha-specific inhibitors, aphidicolin and butylanilinouracil (BuAU), and the identification of clones which carry mutant, drug-resistant pol alpha. The examination of pol alpha structure will attempt to determine: (1) whether pol alpha is a glycoprotein, and (2) whether it is associated with functionally related proteins. If pol alpha is a glycoprotein, the role of carbohydrate in the intracellular synthesis and function of pol a will be studied. The identification of pol alpha-associated proteins will use monospecific, pol alpha-specific antibody and a nuclear DNA replication system.
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