REGULATION OF REPLICATION OF AN ITERON-CONTAINING DNA MOLECULE

This chapter discusses the structure and mechanisms controlling the initiation frequency of plasmid R6K, primarily the γ ori. Because all three origins share many properties, the small γ ori should reveal the most fundamental mechanisms, regulating the replication of the entire 38-kb R6K. Also, the deceptively simple γ ori was chosen for study, because it has all the features needed to elucidate the regulation of replication of iteron-containing DNA molecules in general. Plasmid R6K is a complex replicon, in which the replication forks emanate from any of the three positions called the α, β, and γ origins. All three origins require the R6K-encoded rep protein for their activity. When the α and β sequences are removed, the remaining γ ori can replicate autonomously and the α and β origins require the γ -ori sequence in cis to function. The mechanism, underlying stable inheritance of plasmid R6K, ensures that the plasmid copy-number is maintained at a constant level of 15-20 per chromosomal equivalent. This level can be reduced substantially, by an increase in the intracellular concentration, of two plasmid-encoded elements: the π protein and its binding sites. These two elements have a dual role: they are essential for replication, but can also inhibit origin activity. The negative role of both the elements has also been demonstrated genetically, either by mutating the iterons or by mutating the pir gene. © 1994 Academic Press Inc.