Structural analysis of replicating DNA following exposure to cytotoxic drugs: implications for current models of DNA synthesis in mammalian cells.
The polynucleotide length of single-stranded regions in double-stranded DNA may be determined by caffeine gradient elution from benzoylated DEAE-cellulose. On the basis of this principle, analysis has been made of sheared, deproteinized DNA isolated from synchronized lymphoblastoid cells. Two classes of single-stranded regions were detected. A minor fraction of replicating DNA contained single-stranded regions of 200 nucleotides, whilst the major structural discontinuity involved single-stranded regions of 1-4 kilobases. Newly incorporated [3H]thymidine was principally associated with the latter. Using a 'pulse-chase' protocol, the effect of certain cytotoxic drugs (and related compounds) on the proportion of replicating DNA exhibiting single-stranded character was assessed. The effects were variable. The proportion was increased by hydroxyurea and 3-aminobenzamide, but decreased by inhibitors of DNA polymerase and, to a greater extent, by inhibitors of topoisomerase. Caffeine gradient elution associated drug-induced changes with the radiolabelling of long single-stranded regions. The results are consistent with models of DNA replication involving DNA polymerization remote from replicating forks.
Stewart
B W
BW
Children's Leukaemia and Cancer Research Unit, Prince of Wales Children's Hospital, Sydney, NSW, Australia.
Catchpoole
D
D
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002460:Cell Line; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004247:DNA / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects; D006801:Humans; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000970:Antineoplastic Agents; D004277:DNA, Single-Stranded; D014316:Tritium; D004247:DNA; D013936:Thymidine