AGE-ASSOCIATED CHANGES IN THE TEMPLATE-READING FIDELITY OF DNA-POLYMERASE-ALPHA FROM REGENERATING RAT-LIVER

DNA polymerases (deoxynucleosidetriphosphate : DNA deoxynucleotidyltra nsferase EC 2.7.7.7.) were extracted from regenerating livers from you ng and aged rats. DNA polymerase a was separated and partially purifie d by DEAE-cellulose column chromatography, polyethyleneglycol precipit ation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver of a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [P-32]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleo tides were developed by two dimensional thin layer chromatography on P EI cellulose plates. Spots containing [P-32]dTMP were observed when DN A polymerase from a 24 month-old rat was used, but none was found in p olynucleotides synthesized using DNA polymerase from a 4 month-old rat . Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase alpha. (C) 1996 Elsevier Science Ireland Ltd.