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

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Cellular aging, or senescence, is characterized by a finite number of cell divisions.
• DNA replication fidelity is crucial for maintaining genomic stability during cell division.

Purpose of the Study:

• To investigate changes in DNA polymerase alpha fidelity and 3'-->5' exonuclease proofreading activity during the aging of human diploid fetal lung fibroblasts (TIG-1 cells).
• To determine the relationship between these enzymatic activities and the increase in mutation frequency observed at the end of the cellular lifespan.

Main Methods:

• Isolation and characterization of DNA polymerase alpha and 3'-->5' exonuclease from TIG-1 cells at various population doubling levels (PDL).
• Assay of DNA polymerase alpha fidelity and 3'-->5' exonuclease activity in vitro.
• In vitro DNA synthesis experiments using DNA polymerase alpha and 3'-->5' exonuclease from cells at different PDLs.

Main Results:

• DNA polymerase alpha fidelity remained high until late passage (65-69 PDL), then decreased sharply.
• 3'-->5' exonuclease activity remained stable until 61 PDL, then rapidly declined, reaching ~50% of early passage levels by 69 PDL.
• Supplementing aged DNA polymerase alpha with active 3'-->5' exonuclease from younger cells improved DNA synthesis fidelity in vitro.

Conclusions:

• The decline in proofreading exonuclease activity precedes the sharp drop in DNA polymerase alpha fidelity.
• An imbalance between DNA polymerase alpha activity and 3'-->5' exonuclease proofreading contributes to increased mutation frequency near the end of the cellular lifespan.
• These findings suggest a mechanism for the rise in mutation frequency associated with cellular senescence.