N-acetoxy-N-2-acetylaminofluorene-induced damage on SV40 DNA: inhibition of DNA replication and visualization of DNA lesions

Abstract

The chemical carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), which induces well characterized DNA lesions, strongly inhibits Simian virus 40 (SV40) DNA replication. By using SV40 mutants which were temperature-sensitive for replication initiation, we were able to synchronize SV40 DNA replication and therefore to introduce AAAF-induced lesions only on unreplicating SV40 molecules. One to two acetylaminofluorene (AAF)-adducts per SV40 genome inhibit more than 90% of normal semi-conservative DNA synthesis. SV40 replicative intermediates (RIs) from AAAF-treated infected cells, purified through neutral sucrose gradients and BND-cellulose column, possess a structure different from the usual Cairns molecules found in the untreated cultures. Both by neutral and alkaline sucrose gradients and by electron microscopy, the RIs isolated from treated cells appear as complex molecules with single-stranded portions and sometimes with a tailed structure. Moreover, the newly synthesized strands found in these molecules are equal in size to the average distance between AAF-adducts on the template strand, indicating that AAF-adducts represent a block for the SV40 DNA replication. By using specific anti Guo-AAF antibodies and electron microscopy, we show the presence of an AAF adduct at halted replication forks, i.e. showing a DNA replication block in a mammalian replicon for the first time. We therefore assume that AAF-adducts severely block the progression of the replication forks by inhibiting, at least, the in vivo polymerization of the leading strand.