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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG006347-02
Application #
3117330
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1987-05-01
Project End
1992-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
2
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Type
Schools of Veterinary Medicine
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
Miller, S D; Crouch, E A; Busbee, D L (1997) An accessory protein of DNA polymerase alpha declines in function with increasing age. Mutat Res 374:125-38
Crouch, E; Miller, S; Wilson, V et al. (1997) A DNA polymerase alpha accessory protein exhibits structural and functional similarities to SV40 large tumor antigen. Mutat Res 374:109-23
Srivastava, V K; Schroeder, M D; Miller, S D et al. (1996) Differential expression of DNA polymerase alpha in normal and transformed human fibroblasts. Mutat Res 316:267-75
Schroeder, M; Miller, S; Srivastava, V et al. (1996) An accessory protein enhances both DNA binding and activity of DNA polymerase alpha isolated from normal, but not transformed, human fibroblasts. Mutat Res 316:237-48
Alterman, M A; Carvan, M J; Busbee, D L (1995) Dose-dependent induction of the microsomal monooxygenase system by phenobarbital and 3-methylcholanthrene in the ad libitum and calorie-restricted female rat. Xenobiotica 25:17-26
Alterman, M A; Carvan, M J; Busbee, D L (1994) Ethoxyresorufin and pentoxyresorufin O-dealkylation by hepatic microsomes from female Fischer 344 rats: effects of age and diet. Mech Ageing Dev 77:1-11
Srivastava, V K; Schroeder, M D; Busbee, D L (1993) Characterization of DNA polymerase alpha from untransformed and pSV3.neo-transformed human fibroblasts. Int J Biochem 25:385-95
Srivastava, V; Miller, S; Busbee, D (1993) Immunofluorescent evaluation of DNA repair synthesis using interactive laser cytometry. Cytometry 14:144-53
Srivastava, V K; Miller, S; Schroeder, M D et al. (1993) Age-related changes in expression and activity of DNA polymerase alpha: some effects of dietary restriction. Mutat Res 295:265-80
Srivastava, V K; Schroeder, M D; Miller, S M et al. (1993) A comparison of DNA polymerase alpha from untransformed and SV40-transformed human fibroblasts. Int J Biochem 25:1053-63

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