Abstract
Aphidicolin, a known inhibitor of DNA polymerase alpha, is a potent inhibitor of nuclear DNA synthesis in HeLa cells but has no effect on the replication of mitochondrial DNA. Parallel experiments with mitochondria incubated in vitro also show no inhibition of DNA synthesis by aphidicolin; however, DNA synthesis in these isolated mitochondria is completely blocked by dideoxycytidine triphosphate, which inhibits DNA polymerase gamma but not the alpha polymerase. The replication of mitochondrial DNA therefore requires only one DNA polymerase of the gamma type.
Highlights
Aphidicolin, a known inhibitor ofDNA polymerase a, is a potent inhibitor of nuclear DNA synthesis in HeLa cells but has no effect on the replicatioofnmitochondrial DNA
By separatingandisolating the nuclear and mitochondrial DNA, we have shown that DNA polymerase a is not involved in mitochondrial DNA replication, a process which requires onlythe y polymerase
Aphidicolin was a slft from Dr Hesp (Imperial Chemical Industries, England) andpBR322 DNA was kindly supplied by David Wing, Roche Institute of Molecpellet was dissolved in 0.3 ml of NCS (Amersham) and the radioactivity was determined in a Beckman LS-250 liquid scintillation counter afteraddition of 3 ml of ACS (Amersham) containing 2 ml of glacial acetic acid/liter
Summary
Cells were pulse-labeled for 2 h with [3ZP]orthophosphate(40 pCi/ml) Under these conditions, control cells showed 80 to 85%of the [“Hlthymidine incorporation into DNA when compared to cells cultured in medium with normal commences [2].At present, strand displacementsyntheses of amounts of phosphate. This magnitude are not known to occur in the replication of the host nuclearDNA It was, thereforeo,f interest that DNA polymerase y, identified as the mitochondrialDNA polymerase [3],has been implicated in another caosfestrand displacement synthesis, namely the replication of adenovirus DNA in Isolation of Nuclei and Mitochondria-The cell suspension was chilled on ice, and cells were collected by centrifugation (850 X g,10 min, 4°C) and washed twice with phosphate-buffered saline (Gibco).
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