Reduction of DNA primase activity in aging but still proliferating cells.
The basis of the well-known decline in cell proliferation with increasing passage number of human diploid fibroblast-like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase alpha bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165-173, 1985]. In the present study, the declining percentages of S-phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase alpha and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase-digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase alpha and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrease in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.
Collins
J M
JM
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Chu
A K
AK
D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology / Q000648:ultrastructure; D002470:Cell Survival; D002474:Cell-Free System; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000378:metabolism; D019915:DNA Primase; D006801:Humans; D066298:In Vitro Techniques; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*
D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II