Evidence for the absence of DNA proofreading in HeLa cell nuclei.
[3H]2-Aminopurine deoxyribonucleoside triphosphate and [32P]dATP were added exogenously at equimolar concentrations to washed HeLa cell nuclei both in the presence and absence of cell cytoplasm. The observed ratio of 2-aminopurine/adenine deoxyribonucleotide incorporation into DNA was about 12%, which is consistent with 2-aminopurine misinsertion frequencies measured in cell-free assays, for various DNA polymerases including alpha-polymerase from calf thymus, Escherichia coli polymerase I, and several mutant and wild type bacteriophage T4 polymerases. Based on the 12% 2-aminopurine/adenine misincorporation ratio, we propose that proofreading of replicating DNA is not occurring in HeLa nuclei, and that discrimination against 2-aminopurine incorporation is governed primarily by a 1.1 kcal/mol difference in free energy between 2-aminopurine.thymine and adenine.thymine base pairs rather than by properties attributable to either the mammalian DNA polymerase or HeLa cell nuclear replication apparatus.
D002467:Cell Nucleus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D007700:Kinetics
D004273:DNA, Neoplasm