Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging.

DNA polymerase alpha and the 3'-->5' exonuclease involved in the proofreading of DNA synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase alpha remained high until late passage and fell suddenly just before the end of the life span between 65 and 69 PDL. The activities of the 3'-->5' exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the activity decreased rapidly in more aged cells. The 3'-->5' exonuclease activity at 69 PDL was about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase alpha from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3'-->5' exonuclease from the same cells. However, not all errors were edited out since the ratio of DNA polymerase activity to 3'-->5' exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA synthesis by error-prone DNA polymerase alpha from aged cells was improved by the addition of the highly active 3'-->5' exonuclease from cells at 41 PDL. These results suggested that the mutation frequency rises just before the end of the life span of TIG-1 cells.