Age-associated changes in the template-reading fidelity of DNA polymerase alpha from regenerating rat liver.
DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase alpha was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, 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 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 [32P]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [32P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides 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.
Taguchi
T
T
Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology, Japan.
Ohashi
M
M
D000375:Aging / Q000378:metabolism*; D000818:Animals; D004257:DNA Polymerase II / Q000378:metabolism*; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D009841:Oligonucleotides / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013698:Templates, Genetic
D009841:Oligonucleotides; D004257:DNA Polymerase II