This research proposes to examine the relationship between aging and decreased DNA synthesis associated with DNA excision repair and with mitogen-stimulated blastogenesis in human and mouse cells. Data from an ongoing research program show that DNA pol-a is present in quiescent cells in an inactive form which is activated upon stimulation of the cells to initiate either cell division or DNA excision repair. Data show that inactive DNA pol-a does not bind DNA template/primer, and that initiation of DNA synthesis necessary for either DNA excision repair or mitogen-stimulated cell division does not proceed in the absence of DNA pol-a activation. Inactive DNA pol-a which is treated with phosphatidylinositol (PI), phosphatidylinositol kinase (PIK), and ATP, or phosphatidylinositol-4-monophosphate (PIP) alone, is activated by transfer of a phosphate from PIP to a subunit of the enzyme. Phosphorylated DNA pol-a has a high binding affinity for DNA template/primer, and initiates DNA synthesis. Active DNA pol-a may be inactivated by treatment with alkaline phosphatase. Fibroblasts treated with the carcinogen r-7,t-8-dihydroxy-t-9,10- epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) begin unscheduled DNA synthesis correlated with phosphorylation of DNA pol-a and an increase in specific activity of DNA pol-a. Lymphocytes treated with the mitogen phytohemagglutinin initiate cell division correlated with phosphorylation of DNA pol-a and an increase in specific activity of the enzyme. Activation of DNA pol-a is directly associated with phosphatidylinositol mobilization in stimulated cells, and fibroblasts or lymphocytes deprived of phospholipids do not activate DNA pol-a efficiently. Our data show that both unscheduled DNA synthesis in carcinogen-treated fibroblasts, and blastogenesis in mitogen- stimulated lymphocytes, decrease as a function of increased age in human subjects, and that decreased DNA synthesis correlates with decreased specific activity of DNA pol-a isolated from the cells. We propose to examine the efficiency of DNA pol-a activation in human lymphocytes, in human and mouse fibroblasts, and in mouse hepatocytes as a function of age. We have characterized the primary phosphorylation mechanism which activates DNA pol-a. We will determine the mechanism(s) involved in regulation of that phosphorylation cascade. We will determine the effects of aging in human and mouse cells on both the phosphorylation cascade and the mechanisms of activation of the cascade. We will determine the difference in DNA pol-a activation by phosphorylation in early and late passage fibroblasts from young and old mice and humans, in hepatocytes from young and old mice, and in lymphocytes from young, middle aged, and older humans.
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