Oligomeric Initiator Protein-Mediated DNA Looping Negatively Regulates Plasmid Replication In Vitro by Preventing Origin Melting  Shamsu Zzaman, Deepak Bastia  Molecular Cell  Volume 20, Issue 6, Pages 833-843 (December 2005) DOI: 10.1016/j.molcel.2005.10.037 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Diagrams Showing the Structure of oriF and incC and the Coupling of the Two Elements by Dimeric RepE-Mediated Looping (A) Diagram of the structure of the replication origin of F. The detailed features of the ori have been described in the text. (B) A model showing DNA looping and dimeric RepE-mediated iteron coupling. (C) Filter binding experiment showing the binding of the RepEwt and RepE∗ proteins to labeled DNA probe (20 ng). Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Replication of the Chimeric pZoriF and pZoriF-incC and the pUC19 DNA Templates In Vitro (A) Photograph of a Coomassie blue-stained SDS-polyacrylamide gel of the purified protein components used in the in vitro replication reaction. (B) The two chimeric templates replicated vigorously, whereas the pUC19 did not support more than background levels of incorporation, thus showing that replication was initiated from the F sequence. (C) Replication data showing that ATP was not only needed for chaperone-mediated remodeling of RepEwt protein but also was needed during the second incubation step to sustain replication. Omission of any one of DnaK, DnaJ, or GrpE during the preincubation with RepEWt largely abolished replication during the second incubation step even if ATP was supplied at the second step. The error bars represent the standard deviation from the mean of three independent experiments. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Chaperones on the Oligomeric States of RepE (A-I) Gel-filtration profiles of RepE wt (filled diamonds) and mutant RepE∗ (filled squares) and DNA binding activities of the fractions (shown in red). Note that the monomer readily binds to iteron DNA in a filter binding assay, whereas a shoulder of the wt RepE located in between the main dimeric peak and the known location of the monomeric peak shows some DNA binding activity. (A-II) Gel-filtration profiles of chaperone- treated wt RepE and specific DNA binding activities of the fractions. (B) Gel-mobility-shift analysis of RepEwt, chaperone-treated RepE and monomeric RepE∗, and 10 fmol of labelled, single-iteron DNA probe. (C) A model showing the interpretation of the data. The dimer (blue-red) after chaperone treatment and ATP hydrolysis is remodeled into a premonomer (yellow-red) that folds into the monomeric form (green) upon binding to the iteron DNA. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 A Model of the Mechanism of Negative Regulation of DNA Replication by Iteron Coupling and Experimental Verification of Its Key Predictions (A) Experimental data showing the effect of both pZoriF and pZoriF-incC templates on net replication by challenging the replication reaction with 0–400 ng of dimeric RepEwt protein. Whereas an increasing range of the RepEwt protein caused only a modest and gradual reduction in net synthesis of the oriF template, the oriF-incC template showed a sharp decline in synthesis at ∼50 ng of RepEwt protein and then reached a plateau. (B) Replication of the oriF-incC template DNA that was challenged with an equivalent range of concentrations of the mutant. Monomeric RepE∗ protein showed only a modest and gradual inhibition of replication as a function of the added RepE∗ concentration. (C) Replication of the oriF-incC template preinitiated with the RepE∗ protein was inhibited in the presence of an increasing range of concentrations of Rep Ewt protein, whereas the oriF template showed no significant inhibition when challenged by the same range of concentrations of the dimer. More dimeric protein was needed to inhibit replication initiated with RepE∗ than one initiated by chaperone-treated RepEwt (compare Figure 4A with Figure 4C). (D) Diagram showing the remodeling of RepE dimers into monomers by chaperone-catalyzed ATP hydrolysis and the subsequent binding to iteron DNA. The activated RepE, in the presence of HU and DnaA, causes open complex formation at the oriF that probably involves wrapping of the ori DNA around a core of four monomeric RepE. (E) Addition of dimeric RepE is believed to cause isomerization of the origin complex to a looped and bridged structure that inhibits open complex formation. The error bars represent the standard deviation from the mean of three independent experiments. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Role of the DnaK Chaperone System in Origin Melting (A) Schematic representation of the strategy for the induction and detection of unwinding in the DNA templates by chaperone-treated RepE wt, HU, and DnaA proteins. The melted region was trapped by KMnO4, digested with S1 nuclease, cleaved with the indicated restriction enzymes, and end labeled. (B) Autoradiogram of the reaction products prepared under various conditions and resolved in 1.6% agarose gels. Abbreviations are as follows: Ewt, RepEwt; A−, DnaA minus; Chp−, Chaperone minus; E∗, monomeric RepE∗ mutant form. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Inhibition of Origin Melting by Molar Excess of RepEwt Dimeric Protein RepEwt protein was first preincubated with DnaJ, DnaK, GrpE, and ATP and subsequently incubated with DnaA, HU, and a range of concentrations (0–60 ng, 0–1 pmol) of RepEwt that was not pretreated with chaperones. (A) Autoradiogram (left panel) shows the melting reaction of the oriF template (without incC) as a function of RepEwt concentration (0–1 pmol). The intensities of the fragments III and IV that were generated by cleavage of the melted region in fragment II were not inhibited by dimeric RepEwt protein. In contrast, similar manipulations of the oriF-incC template resulted in a progressive reduction of the intensities of fragments III and IV as a function of the same range of concentrations of RepEwt, indicative of inhibition of ori melting (right panel). (B) Autoradiogram showing the effect of the addition of monomeric RepE∗ on ori melting. RepE∗ did not reduce the intensities of fragments III and IV in the pZoriF (left panel) or in the pZoriF-incC DNA (right panel). Lanes 1–6 in each panel represent the additions of 0, 20, 30, 40, 50, and 60 ng of either RepEwt or RepE∗ proteins as indicated. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Measurement of incC-oriF Looping in the Presence of Various Forms of RepE Proteins (A) A model based on the crystal structure of monomeric RepE∗ bound to a single iteron. The monomeric form has two regions of DNA binding (X and Y) and almost symmetrical N- and C-terminal halves. Dimerization apparently occludes the DNA binding domain of the protein that contacts the 3′ end of the iteron. The dimer is postulated to bridge pairs of iteron bound monomers in the looped structure by contacts at the so-called weak interaction sites; the dimerization domain represents the strong protein-protein interaction surface. (B) The strategy of the ligation experiments to measure DNA looping. The EcoR1-generated ends were 5′ end labeled, and 20 ng (20 fmol) of the labeled DNA was incubated either with no protein or with the monomeric form, the dimeric form, or with the dimeric form after prebinding of the DNA to the monomeric form. The DNA-protein complexes (or naked DNA control) were incubated with a limiting amount of T4 DNA ligase at 12°C for 60 min and digested with gene 6 exonuclease. The DNA was then precipitated, dried on filters, and counted. The internalization of the terminal label caused by ligation is a measure of the rate of ligation, which in turn is a measure of the DNA looping that brought the two ends into close proximity. (C) The labeled DNA substrate was prebound to an excess of monomers (3.4 pmol) and then challenged separately with the range of concentrations of monomer and dimers, ligated for a range of time periods, digested with gene 6 exonuclease to the limit, and the exonuclease resistant counts were measured. (D) Ligation-exonuclease experiments in which the time of ligation was kept fixed and the concentrations of the various forms of RepE were varied. The DNA (20 ng) was first incubated with enough monomers to yield a complete gel shift and then was incubated with the range of concentrations of dimers or monomers (control). After addition of ligase, further incubation was continued for 60 min at 12°C followed by digestion with gene 6 exonuclease. The surviving DNA was TCA precipitated on glass-fiber filters, washed with ethanol, dried, and counted. Note that a combination of monomers and dimers, as predicted by the looping model ([A] and [B]), yielded the highest rate of ligation of the DNA ends. The error bars represent the standard deviation from the mean of three independent experiments. Molecular Cell 2005 20, 833-843DOI: (10.1016/j.molcel.2005.10.037) Copyright © 2005 Elsevier Inc. Terms and Conditions