We propose to establish the expression of an adult form of DNA polymerase alpha as a biomarker of aging in rodent model systems, and to determine whether dietary restriction can alter the age-related progressive expression of the adult form of this enzyme. Data from an ongoing research project show that DNA polymerase alpha isolated from human cells in vitro, or from mouse tumor cells grown in vitro or as a solid tumor, have two forms of DNA polymerase alpha. One enzyme form is indistinguishable from polymerase alpha purified from fetal- derived fibroblasts. This isozyme, which is highly active, declines as a present of total polymerase protein with increased age of the cell donor. A second isozyme, which has low activity but can be transiently activated by interaction with a product of the phosphoinositide cascade, increases as a percent of total polymerase protein with increased age of the cell donor. These isozymes of DNA polymerase alpha purified from either human or mouse cells can be separated because they differ in charge, in their affinity of binding to an anti-DNA polymerase alpha monoclonal IgG specific to what we call the polymerase regulatory subunit, and in their affinity of binding to enzymatically gapped DNA template/primer. The fetal-type isozyme of DNA polymerase alpha exhibits about a 10-fold greater specific activity than is shown by adult-type enzyme, and can not be further activated. The adult-type isozyme of DNA polymerase alpha has low specific activity, but can be transiently activated by interaction with inositol-1,4-bisphosphate, a hydrolysis product of phosphatidylinositol-4-monophosphate. Activated adult-type polymerase alpha can not be distinguished from the fetal form of the enzyme. The progressive increase in expression of adult-type DNA polymerase alpha with increased age of the cell donor is quite significant, with fetal cells showing 0% of the adult enzyme form, and cells derived from a 66 year old male exhibiting 94% of the total polymerase protein as adult-type enzyme. We will characterize the expression of DNA polymerase alpha fetal vs adult forms as a function of age in mouse and rat model systems, and in the F1 hybrids of the respective rat and mouse strains. We will determine whether dietary restriction alters the progressive decline of fetal enzyme expression and the progressive increase in adult enzyme expression as a function of increased age of the animals. We will compare DNA polymerase alpha expression as an aging biomarker in the rodent systems with DNA polymerase alpha expression as an aging biomarker in human fibroblasts to assess the potential for future use of this enzyme system in human biomarker studies.
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