title: Age-related changes in DNA polymerase alpha expression.
abstract: DNA polymerase alpha isozymes differing in specific activity and affinity of binding to DNA were purified from human fibroblasts derived from donors of different ages. Fetal-derived fibroblasts expressed a single, high-activity enzyme (A2), with high affinity of binding to DNA. Adult-derived fibroblasts exhibited two forms of DNA polymerase alpha, one identical to the fetal enzyme, and a second with about tenfold less activity showing low affinity of binding to DNA (A1). The ratio of DNA polymerase A2/A1 decreased dramatically with age, from 100% A2 in fetal-derived fibroblasts to about 94% A1 in fibroblasts derived from a 66-year-old donor. The DNA binding affinity of polymerase alpha A1 from adult-derived fibroblasts increased concomitant with a significant increase in activity when the enzyme was treated with phosphatidylinositol-4-monophosphate (PIP), or with inositol-1, 4-bisphosphate (I(1,4)P2). The enzyme reverted back to a less active form, with loss of the noncovalently bound I(1,4)P2, as a function of time. When permeabilized human fibroblasts with low DNA excision repair capacity were treated with 7,8-dihydrodiol-9,10-epoxybenzo(a)-pyrene (BPDE) in the presence of 32P-ATP, phosphatidylinositol, and cycloheximide, excision repair was initiated and 32P-labeled DNA polymerase alpha was recovered in the absence of de novo protein synthesis. DNA synthesis associated with either scheduled DNA synthesis or BPDE-initiated excision repair declined as a function of increased age in human cells. The data suggest that the decline in both DNA excision repair-associated and mitogen-activated DNA synthesis may be correlated with decreased total intracellular levels of DNA polymerase and with the decline in polymerase alpha activity as a function of age, that DNA repair-associated initiation of DNA synthesis in adult-derived cells may increase with activation of a pool of low activity DNA polymerase alpha, and that DNA polymerase alpha activity increases as a function of enzyme interaction with a component of the PI phosphorylation cascade.
authors.affiliation: Department of Anatomy, College of Veterinary Medicine, Texas A&M University, College Station 77843.
authors.forename: D
authors.initials: D
authors.lastname: Busbee
authors.forename: V
authors.initials: V
authors.lastname: Sylvia
authors.forename: G
authors.initials: G
authors.lastname: Curtin
authors.forename: S
authors.initials: S
authors.lastname: Peng
authors.forename: V
authors.initials: V
authors.lastname: Srivastava
authors.forename: R
authors.initials: R
authors.lastname: Tilley
mesh_terms: D002470:Cell Survival / Q000502:physiology; D002845:Chromatography; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000378:metabolism*; D004101:Dihydroxydihydrobenzopyrenes / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007295:Inositol Phosphates / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology
chemical_list: D004101:Dihydroxydihydrobenzopyrenes; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D004247:DNA; D004257:DNA Polymerase II