Mechanism of action of the Rep protein from the Dictyostelium Ddp2 plasmid family.

The two-hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers when expressed in yeast cells. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously expressed and purified Ddp2 Rep protein showed that it is a DNA binding protein. The nucleosomal organization of Ddp2 and Ddp6 in their inverted repeat and promoter regions was investigated. Analysis of mutants derived from the Ddp6 plasmid revealed that its Rep protein is required for nucleosome positioning (i.e., phasing) to occur in the promoter region. On the other hand, nucleosome positioning in the inverted repeat regions of both plasmids is not dependent on Rep protein but on either a feature of the DNA sequence or the binding of cellular factors, perhaps the Dictyostelium origin recognition complex. Rep protein is likely involved in transcription regulation and control of DNA replication, specifically amplification of plasmid at low copy numbers. The formation of homomultimers may be required for their regulatory activity.