Accumulation of 10-kilobase DNA replication intermediates in cells treated with 3-aminobenzamide

Abstract

During eukaryotic DNA synthesis there is formation of, in addition to Okazaki fragments, discrete 10-kilobase (kb) DNA replication intermediates. We have investigated the ligation of 10-kb DNA replication intermediates to high molecular weight DNA, using the drug 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase. In human melanoma cells treated with this inhibitor, there is an accumulation of 10-kb DNA. In contrast, in cells treated with aphidicolin, which inhibits DNA polymerase alpha, there is continued ligation of 10-kb DNA to high molecular weight DNA. Furthermore, using sequential treatment with aphidicolin and 3-aminobenzamide, one can observe the conversion of radiolabeled Okazaki fragments into 10-kb intermediates. The 10-kb DNA pieces are, however, not ligated to high molecular weight DNA in the presence of 3-aminobenzamide. Our results imply that functioning poly(ADP-ribose) synthetase is necessary for the ligation process.