Changes in DNA polymerases .alpha., .beta., and .gamma. during the replicative life span of cultured human fibroblasts

Abstract

DNA polymerases from IMR-90 human diploid fibroblasts at various passage levels and from HeLa cells were purified and fractionated into .alpha.1, .alpha.2, .alpha.3, .beta., and .gamma. species and subspecies, and then the accuracy with which each one copied synthetic template-primers was measured in the presence of Mn2+ or Mg2+. All activities from fibroblasts of later population doubling levels incorporated noncomplementary triphosphates more frequently than did the same polymerase type from earlier population doubling levels. HeLa polymerase activities copied several different templates in the presence of Mn2+ with greater fidelity than enzymes from fibroblasts of population doubling level 27 or greater. The total DNA polymerase activity extracted from IMR-90 cells decreased with increasing population doubling levels. The .alpha.-polymerase activity generally declined with increasing population doubling levels, while .beta.-polymerase activity remained relatively constant, except at the very end of the cellular replicative life span. In addition, the amounts of .alpha.2 and .alpha.3 became progressively lower relative to .alpha.1, and a new .alpha.-type polymerase activity, .alpha.0, appeared upon DEAE-cellulose chromatography. HeLa cells also contained 3 .alpha. species, though 2 of them eluted from DEAE-cellulose at higher phosphate concentrations than .alpha. species from fibroblasts. Postconfluent IMR-90 cells of population doubling level 21 had a decreased level of .alpha.-polymerase relative to that recovered from rapidly growing cells. This polymerase activity had some chromatographic properties similar to enzyme from late-passage cells. In addition, the .alpha.-, .beta.-, and .gamma.-, and .gamma.-polymerases from these cells had decreased fidelities relative to those isolated from subconfluent cells.