π Protein- and ATP-dependent transitions from ‘closed’ to ‘open’ complexes at the γ ori of plasmid R6K

R6K-encoded π protein can bind to the seven, 22 bp tandem iterons of the γ origin. In this work, we use a variant of π, His-π·F107S, that is hyperactive in replication. In vitro, His-π·F107S-dependent local DNA melting (open complex formation) occurs in the absence of host proteins (IHF/HU or DnaA) and it is positioned in the A + T-rich region adjacent to iterons. Experiments described here examine the effects of ATP, Mg(2+) and temperature on the opening reaction. We show that the opening of the γ origin can occur in the presence of ATP as well as AMP-PCP (a non-hydrolyzable ATP analog). This suggests that, for γ origin, ATP hydrolysis may be unnecessary for open complex formation facilitated by His-π·F107S. In the absence of ATP or Mg(2+), His-π·F107S yielded data suggestive of distortions in the iteron attributable to DNA bending rather than DNA melting. Our findings also demonstrate that ATP and π stimulate open complex formation over a wide range of temperatures, but not at 0°C. These and other results indicate that ATP and/or Mg(2+) are not needed for His-π·F107S binding to iterons and that ATP effects an allosteric change in the protein bound to γ origin

Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L(∧)F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A(∧)M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ ori system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in cis competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in trans