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Properties of DNA polymerases from young and ageing human fibroblasts
Abstract:
A comparison was made between two methods of isolating DNA polymerases from MRC-5 fibroblasts. The first method produces DNA polymerase-alpha with a lower molecular weight and other properties that are not normally found for this enzyme. It was concluded that this method produces proteolytically degraded DNA polymerase-alpha. A second method was developed which produces DNA polymerase-alpha with all the normal properties of this enzyme. The specific activity of DNA polymerase was reduced in senescent MRC-5 fibroblasts approximately 2--4-fold. DNA polymerase-alpha accounts for 95% of polymerase activity in young cells and its specific activity during the fibroblast lifespan correlates with the declining cellular growth rate. DNA polymerase-beta is present at 0.3-3% of total cellular activity and its specific activity does not correlate with cellular growth rate. DNA polymerase-gamma accounts for 5% of the polymerase activity in young cells and 20% in old cells. However, the specific activity of the polymerase-gamma is constant throughout the lifespan of MRC-5. The 5 S DNA polymerase-alpha has an increased in vitro error frequency (average 3.6) compared to the 7 S polymerase-alpha. In addition the proportion of 5 S polymerase-alpha rises from 7% in young cells to 29% in senescent cells in an apparently exponential fashion.
Insights
Researchers developed a superior method for isolating DNA polymerase-alpha from fibroblasts. This enzyme
Area of Science:
- Molecular Biology
- Cellular Aging
- Enzymology
Background:
- Fibroblast aging involves changes in DNA replication and repair mechanisms.
- DNA polymerases are crucial enzymes for DNA replication and repair.
- MRC-5 fibroblasts are a commonly used model for studying cellular senescence.
Purpose of the Study:
- To compare two methods for isolating DNA polymerases from MRC-5 fibroblasts.
- To investigate the changes in DNA polymerase activity and properties during fibroblast senescence.
- To characterize different forms of DNA polymerase-alpha and their error frequencies.
Main Methods:
- Isolation of DNA polymerases using two distinct protocols.
- Biochemical characterization of isolated DNA polymerases, including molecular weight and specific activity.
- Quantification of different DNA polymerase activities in young and senescent fibroblasts.
- Assessment of in vitro error frequency for different forms of DNA polymerase-alpha.
Main Results:
- A novel isolation method yields intact DNA polymerase-alpha, unlike a method producing a degraded form.
- Specific activity of total DNA polymerase decreases 2-4 fold in senescent fibroblasts.
- DNA polymerase-alpha activity declines with fibroblast lifespan, correlating with reduced growth rate.
- A smaller form of DNA polymerase-alpha (5S) increases in senescent cells and exhibits higher error frequency.
Conclusions:
- The developed isolation method is superior for obtaining functional DNA polymerase-alpha.
- Cellular senescence in MRC-5 fibroblasts is associated with reduced DNA polymerase-alpha activity and increased error-prone forms.
- Changes in DNA polymerase-alpha may contribute to age-related genomic instability.