Cauliflower Mosaic Virus replication complexes: characterization of the associated enzymes and of the polarity of the DNA synthesized in vitro

Abstract

The synthesis of both strands of CaMV-DNA has been studied in vitro using viral replication complexes obtained by hypotonic extraction of infected plant organelles. Hybridization of the DNA synthesized in vitro to single stranded CaMV DNA probes cloned in bacteriophage M 13 confirmed that the 35 S RNA served as a template for the synthesis of the (-) DNA strand. The response of CaMV DNA synthesis to various inhibitors suggests that a single enzyme directs both steps of the replication cycle. A comparative activity gel analysis of the DNA polymerases present in nuclear extracts from healthy and CaMV-infected turnips revealed an increase of a DNA polymerase species migrating in the 75 Kd range in infected tissue. When the enzyme activity associated with the isolated replicative complexes was similarly analyzed, the 75 Kd polymerase was markedly predominant, confirming that DNA polymerases of the α-type (MW in the 110 Kd range) are not involved in the aphidicolin-insensitive CaMV DNA replication. It seems therefore increasingly probable that CaMV codes for its own polymerase.