Properties of DNA polymerases from young and ageing human fibroblasts.

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.