Altered nuclear deoxyribonucleic acid alpha-polymerases in senescent cultured chick embryo fibroblasts.

Abstract

DNA alpha-polymerase has been partially purified from nuclei of cultured chic, fibroblasts and separated on phosphocellulose columns into two distinct activities designated DNA polymerases alpha(a) and alpha(b), respectively. The enzyme preparations were devoid of activities of DNA beta,gamma-polymerases terminal deoxyribonucleoside transferase, DNase, DNA-dependent RNA polymerase, and phosphatase. DNA polymerases alpha(a) and alpha(b) both having molecular weights of 160 000, constitute 35-50 and 65-50%, respectively, of the activity of alpha-polymerase in the nucleus. These enzymes differ in their requirements for maximal activity, their relative ability to copy oligo(dG)-poly(dC), their response to ribonucleoside triphosphates, and their kinetics of heat inactivation. When the properties of alpha polymerases derived from early or late passage cultures have been compared, no difference could be detected as a function of cell age in the specific activities of the polymerases in crude cell extracts, their chromatographic behavior on diethylaminoethylcellulose and phosphocellulose columns, and their relative abilities to utilize single deoxyribonucleoside triphosphates with activated DNA template. On the other hand, both enzymes become partially heat labile in aging cells. Also, the activity of DNA polymerase alpha(a) from young cells was stimulated by 2--10 mM adenosine or cytidine triphosphates, whereas the same enzyme from old cultures was inhibited by these agents. Conversely, these ribonucleoside triphosphates inhibited the activity of polymerase alpha(b) in young cells but slightly stimulated this enzyme derived from senescent fibroblasts. In addition, the relative ability of DNA polymerase alpha(a) to copy oligo(dG)-poly(dC) decreased in aged cells, whereas that of DNA polymerase alpha(b) increased. We have also observed significant differences in the effects of potassium chloride and N-ethylmaleimide on the activity of DNA polymerase alpha(a) from old cells as compared to young cells. These age-related alterations in the properties of the two avian DNA polymerases may reflect structural or conformational changes in these enzymes.