Mechanism of origin activation by monomers of R6K-encoded pi protein

Abstract

One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.

Figure 1

Roles of π monomers and dimers in the regulation of replication from γ ori. The seven iterons of γ ori are indicated by tandem arrows while the operator/promoter region is represented by two inverted half arrows. Small vertical arrows indicate the start sites for leading strand synthesis. π, encoded by the pir gene, can bind to an iteron as a monomer (crescent) or dimer (double crescent) although the predominant form in solution is dimer. π binds to the operator/promoter only as a dimer. Shading indicates that the two monomer subunits of a dimer make head-to-head contacts while two monomers bound to two tandem iterons are proposed to make head-to-tail contacts. A monomer contacts the iteron with two domains while a dimer contacts the iteron with only one domain of one of the subunits.

Figure 2

In vivo binding assay. Plasmids, oris and genes are labeled. Expression of pir from the P_BAD promoter is arabinose-inducible. cat encodes chloramphenicol acetyl transferase, conferring resistance to cam. bla encodes β-lactamase, conferring resistance to pen. Crescent-shaped symbols represent π monomers.

Figure 3

π binds cooperatively to two iterons in vivo. Experiments were conducted per Figure 2 and Materials and methods. The numbers of transformants on plates with cam+pen were expressed as ratios against the pUC9 control (no iteron). → represent iterons and * represents the G/C⁷>A/T mutation. ‘5’ and ‘10’ represent the number of base pairs between the two iterons. Decreasing ratios suggest increased titration of π by the incoming iteron-containing pUC9 derivative. The data represent the average of three independent experiments.

Figure 4

π●wt, π ●P106L^F107S and π ●M36A^M38A have different binding patterns with a 2-iteron probe. Lane 1 is DNA only. Gray and white triangles represent increasing concentrations of π ●wt and π●P106L^F107S, respectively, starting with 6.25 ng and doubling for each lane. The black square represents 200 ng of π ●M36A^M38A. Black crescents represent π monomers and gray double crescents represent π dimers.

Figure 5

π binds cooperatively to two thermodynamically identical iterons in vitro. Cooperative interactions are influenced by helical rotation and distance between the two iterons. (a) A depiction of each iteron-containing probe, not including flanking DNA. ‘5’ and ‘10’ refer to the number of bp between iterons. ‘1’ and ‘2’ refer to the first and second iterons of γ ori, respectively. (b) Gel shift titrations of purified π with the corresponding probes in (a). Lane 1 is DNA only. White triangles represent increasing concentrations of π ●P106L^F107S, starting with 6.25 ng and doubling for each lane. The black square represents 200 ng of π ●M36A^M38A. Black crescents represent π monomers and gray double crescents represent π dimers. (c) Quantification of gel shift titration data with π ●P106L^F107S. The fraction of the total radioactivity as free DNA (circles), DNA containing a single π monomer (squares), and DNA containing two π monomers (diamonds) was determined. Continuous, dashed, and dotted lines correspond to the best fit of the data for equations 1a–1c, respectively. The protein concentrations in (b) are a subset of those plotted in (c).

Figure 6

The binding affinity of the 1-iteron and 1-iteron* probes. (a) A depiction of each iteron-containing probe, not including flanking DNA. * indicates the G/C⁷>A/T mutation. (b) Gel shift titrations of the binding of purified π to the corresponding probes in (a). Lane 1 is DNA only. White triangles represent increasing concentrations of π ●P106L^F107S, starting with 6.25 ng and doubling for each lane. The black square represents 200 ng of π ●M36A^M38A. Black crescents represent π monomers and gray double crescents represent π dimers. (c) Quantification of gel shift titration data with π●P106L^F107S. The fraction of the total radioactivity as free DNA (circles) and DNA containing a single π monomer (squares) was determined. Continuous and dashed lines correspond to the best fit of the data for Eq. (3). The protein concentrations in (b) are a subset of those plotted in (c).

Figure 7

π binds cooperatively to two heterogeneous iterons in vitro. Cooperative interactions are influenced by helical rotation and distance between the two iterons. (a) A depiction of each iteron-containing probe, not including flanking DNA. ‘5’ and ‘10’ refer to the number of bp between iterons. ‘1’ and ‘2’ refer to the first and second iterons of γ ori, respectively. * represents the G/C⁷>A/T mutation in iteron #2. (b) Gel shift titrations of purified π with the corresponding probes in (a). Lane 1 is DNA only. White triangles represent increasing concentrations of π ●P106L^F107S, starting with 6.25 ng and doubling for each lane. The black square represents 200 ng of π ●M36A^M38A. Black crescents represent π monomers and gray double crescents represent π dimers. (c) Quantification of gel shift titration data with π ●P106L^F107S. The fraction of the total radioactivity as free DNA (circles), DNA containing a single π monomer (squares), and DNA containing two π monomers (diamonds) was determined. Continuous, dashed, and dotted lines correspond to the best fit of the data for equations 1a–1c, respectively. The protein concentrations in (b) are a subset of those plotted in (c).

Figure 8

π M36A^M38A binds without cooperativity to a two-iteron probe in vitro. (a) Gel shift titrations of purified π with the 2-iteron probe. Lane 1 is DNA only. The black triangle represents increasing concentrations of the dimer-biased π variant π ●M36A^M38A: 12.5 ng, 25 ng, 50 ng, 100 ng, 150 ng, 200 ng, and 250 ng. Gray and white squares represent 200 ng of π ●wt and π ●P106L^F107S, respectively. Black crescents represent π monomers and gray double crescents represent π dimers. (b) Quantification of gel shift titration data with π ●M36A^M38A. The fraction of the total radioactivity as free DNA (circles), DNA containing a single π dimer (squares), and DNA containing two π dimers (diamonds) was determined. Continuous, dashed, and dotted lines correspond to the best fit of the data for equations 1a–1c, respectively.