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Mech Ageing Dev. 1996 Dec 20;92(2-3):143-57.

Age-associated changes in the template-reading fidelity of DNA polymerase alpha from regenerating rat liver.

Author information

1
Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology, Japan.

Abstract

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.

PMID:
9080395
[Indexed for MEDLINE]

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