An accessory protein enhances both DNA binding and activity of DNA polymerase α isolated from normal, but not transformed, human fibroblasts

Abstract

DNA polymerase α/primase (pol α) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol α from either fetal-derived fibroblasts (W138), or pSV3.neo-transformed GM3529 fibroblasts. The pol α specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol α isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol α from GM3529 cells. GM3529T pol α was immunoreactive with both anti-pol α and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol α accessory protein, αAP, isolated from L1210 cells. Pol α from GM3529T cells showed no increase in activity in the presence of αAP, while pol α isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with αAP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol α. In the presence of pol α from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of αAP resulted in an even greater decrease in DNAm mobility. These data indicate that αAP increased pol α binding to SV40 dsDNA, or that αAP bound the DNA in addition to previously bound pol α. GM3529 pol α also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol α with associated TAg did not bind the non-viral dsDNA unless αAP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol α from cells derived from aged human donors. We suggest that a decrease in endogenous αAP interaction with pol α may account, in part, for the loss of DNA binding affinity and specific activity of pol α from GM3529 cells derived from an aged donor.