Augmented nuclease activity during cellular senescence in vitro.
The molecular correlates of the limited proliferative potential of normal human diploid fibroblasts and extensive single-strand breaks in the genomic DNA of these cells were examined by transfection analyses in which DNA replication could be uncoupled from DNA damage and repair. Both supercoiled (fmI), and restriction endonuclease-cleaved, linear (fmIII) molecules of a well-defined bacterial plasmid DNA, pBR322, were transfected into, and subsequently recovered from, early and late passage fibroblasts. Southern blot analysis revealed that fmI DNA was converted by random nicks into fmII DNA slightly more rapidly in late passage cells compared with cells at early passage. Similarly, fmII and fmIII DNAs also sustained multiple random nicks and no appreciable net religation of free ends of fmIII DNA could be detected at either passage. In addition, the efficiency of in vitro ligation of fmIII DNA recovered from late passage cells was also reduced, compared with that from early passage cells, as determined by Southern blotting. These data suggest that in the absence of DNA replication, a putative nuclease activity may contribute to DNA damage observed in senescent cells, which, in turn, may be causally related to their limited replicative potential.
Dayton
M A
MA
Department of Medicine, Indiana University School of Medicine, Indianapolis 46223.
Nahreini
P
P
Srivastava
A
A
D015139:Blotting, Southern; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial; D003851:Deoxyribonucleases / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D014162:Transfection
D004269:DNA, Bacterial; D004247:DNA; D003851:Deoxyribonucleases