Repair of acetyl-aminofluorene modified pBR322 DNA in Xenopus laevis oocytes and eggs; effect of diadenosine tetraphosphate

Abstract Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DNA repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coli . The repaired DNA is even efficiently replicated, the egg being “activated” by the microinjection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase α. However, the repair appears to be very limited, since it does not respotre the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and/or to a very low level of DNA polymerase β. This system was used to study the effect of diadenosine tetraphosphate (Ap 4 A) on DNA repair. Ap 4 A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap 4 A could be involved in switching off the replication machinery when DNA is badly damaged, thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.