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

Abstract

DNA polymerase α 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 α 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 α 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 α 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.