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Journal of Bacteriology, July 2007, p. 4953-4956, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00109-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the /DNA Interface

Selvi Kunnimalaiyaan, Sheryl A. Rakowski, and Marcin Filutowicz^*

Department of Bacteriology, University of Wisconsin, 420 Henry Mall, Madison, Wisconsin 53706

Received 19 January 2007/ Accepted 13 April 2007

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In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of protein monomers and dimers. Here we investigate the following six amino acids of , encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.

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Rep proteins activate replication origins (ori's) by binding to tandem repeats in the DNA called iterons (3, 8, 13). When the sequences of iterons and rep genes are compared across families, striking similarities are observed (2, 10, 16-18). A breakthrough in Rep/iteron studies came when Komori et al. published the crystal structure of a RepE monomer (from the F plasmid) in complex with its iteron DNA (10). The structure revealed that the Rep monomer contains two DNA-binding domains, an N-terminal WH1 (winged helix 1) and a C-terminal WH2 domain, with both domains used for iteron binding. A sequence alignment of several Reps presented by our group (10, 13) is a useful reference for identifying possible conserved amino acids in replication initiators, including RepE and the protein (described below). Based on the homologies observed in this class of proteins, Sharma et al. subsequently reported theoretical three-dimensional models for several Reps (18). The accuracy of these models remained to be tested.

In a minimal R6K replicon, an ori called is activated by the Rep protein, , which is encoded by the pir gene (Fig. 1A) (4, 6, 7, 9, 15). exists in both monomeric and dimeric forms, with monomers being activators of replication and dimers functioning both as replication inhibitors and as autorepressors of pir. The latter function is mediated by dimers binding to inverted repeats in the promoter that share sequence similarity with the iterons. monomers show little to no affinity for the inverted repeats but bind to an iteron better than dimers do (20). Insights into how achieves these similar but functionally distinct interactions were sought from analyses of Rep structure and the contacts that occur at the protein/DNA interface.

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FIG. 1. Elements of a minimal R6K replicon. (A) Roles of protein in the regulation of ori. The protein, encoded by the pir gene, exists in two forms, i.e., monomers (shaded dark gray) and dimers (shaded light gray). Monomers activate replication (i), dimers inhibit replication (ii), and dimers also bind to the inverted repeats to repress pir transcription (iii). binding sites are represented by black arrows. The seven iterons of ori, also called direct repeats, are indicated by tandem arrows, whereas a pair of shorter inverted arrows indicates the inverted repeats in the pir operator/promoter. There is an eighth iteron just upstream of the inverted repeats. The sequence of iteron 1 is also shown, and thymine 27 is marked in bold. (B) Amino acids of selected for mutational analysis. A model of a monomer bound to a ori iteron (14), with the side chains of six amino acids targeted by site-directed mutagenesis, is shown in a space-filling form. The N-terminal and C-terminal domains of are shown in white and green, respectively. DNA strands are represented in purple (top) and gray (bottom). Some of the iteron positions believed to be important for monomer binding are shown as spheres on the DNA strand.

In previous work from this lab, we determined the importance of individual bases in the iteron for the binding of monomers and dimers (14). Beyond this, we knew of no other experimental data addressing the contact residues of protein and DNA; a solved /iteron cocrystal structure was not available at that time. In the present study, we investigated the protein/DNA interface again, this time with an interest in identifying amino acids of that contact DNA. By analyzing the theoretical structural model of the monomer/iteron complex, we identified approximately 2 dozen amino acids as candidates for DNA contact and narrowed the list down to six for further study (Fig. 1B). Four (Ser71, Gly131, Arg225, and Arg254) appeared to be positionally conserved within the RepE/iteron cocrystal structure (Ser75, Gly125, Arg206, and Arg233, respectively). Tyr74 was strongly suspected to contact a thymine residue in the half of the iteron bound only by monomers (14). Lastly, Gly211 was of interest because it falls in an unstructured loop region positioned at the /DNA interface; this region appears to be unique among modeled Rep proteins, and its function, if any, is unknown. We hypothesized that these amino acids might be important for DNA binding and, consequently, the regulatory control of DNA replication. To test this, the six amino acids were changed (individually) via site-directed mutagenesis to alanines, and the resulting variants were characterized in vivo and in vitro.

variants display altered replication control in vivo. Each of the alanine substitution variants of was tested for the ability to support the replication of a "suicide" ori plasmid, pFW25 (22), that does not encode its own protein. Plasmids expressing wild-type (wt) (pJWW801) (12) and variant proteins carried pir genes under the control of a T7 promoter. The plasmids were transformed into Escherichia coli BL21(DE3), which carries an IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible T7 RNA polymerase gene in the chromosome. Western blots were done to confirm the expression of the variants (Fig. 2 and data not shown). As expected, although IPTG induction did elevate levels, detectable levels of the protein were also observed in the absence of IPTG due to "leaky" transcription. Plasmid-bearing BL21(DE3) strains that were grown in the absence of IPTG were made competent and used in transformation experiments with pFW25 DNA. We observed that the control strain producing wt (from pJWW801) yielded colonies on selective medium (for pFW25) whether or not pir expression was induced by adding 0.1 mM IPTG to the plates. In contrast, when wt pir (in pJWW801) was replaced by an alanine substitution mutant, repeated attempts to establish pFW25 were unsuccessful for five of the six variants tested; only the G211A mutant produced colonies. As noted above, amino acid 211 falls in a unique unstructured loop region of the protein whose function cannot be inferred by homology modeling. These results are unlikely to be attributable to variation in the protein expression levels of our constructs for two reasons. First, repeated Western blots failed to demonstrate reproducible differences in expression. Second, even if the variation observed had been reproducible, for each variant at least one level of expression (induced and/or uninduced) fell within the range of wt expression that allowed successful establishment of the ori plasmid (Fig. 2).

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FIG. 2. Western analysis. Cells containing pJWW801 (producing wt ) and derivative plasmids producing variants were grown in the absence (–) or presence (+) of IPTG (0.1 mM). Samples (0.1 ml) of cells standardized to an optical density at 600 nm of 1.2 were lysed and subjected to Western analysis using anti- antibodies. A sample of purified wt protein was loaded as a control (lane C).

In addition to being a replication initiator, can also inhibit replication (5). To test the inhibitor activity of the six variants, we performed another two-plasmid transformation assay, similar to the one described above except that the suicide plasmid was replaced with a complete ori replicon that encodes its own wt protein (pFL129) (21). The wt -producing pJWW801 plasmid is compatible with the pFL129 ori replicon when low levels of protein are produced. However, the overproduction of wt inhibits the replication of ori plasmids (e.g., pFL129), presumably due at least in part to increased competition for iteron binding by dimers (13). Using this in vivo system, we compared the inhibitory activities of variants to the activity of wt (Fig. 3). The data show that when wt or the G211A mutant was overproduced, the number of transformants that exhibited the antibiotic resistances of both plasmids was reduced approximately 50% compared to that for the uninduced control. In contrast, when any of the other variants was overproduced, the number of colonies was reduced >90%. This observation suggests that these variants inhibit replication more strongly than does wt . The ability of a related assay to determine the relative strength of replication inhibition has been attributed to several factors (1). These factors include the facts that the level of chloramphenicol resistance produced by the ori construct is dependent on the plasmid copy number and that the copy numbers of individual cells can vary in a population. Notably, in both in vivo assays, conditions that reduced the number of colonies (inhibited ori plasmid replication) also led to reduced colony sizes (1; this work and unpublished observations). Taken together, the positive and negative functional assays indicate that five of the six amino acids of under study contribute to DNA replication control, consistent with the predicted roles for these residues in iteron binding.

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FIG. 3. Replication inhibitor function of variants (in vivo). DNA from the ori plasmid pFL129 was added to competent cells containing pJWW801 (producing wt ) or derivative plasmids producing variants (grown in the absence of IPTG). Transformation mixtures were plated on selective medium lacking IPTG (uninduced) or induced with 0.1 mM IPTG to overexpress variants (including wt ). The histogram shows the numbers of colonies obtained when the cells were uninduced (light shading) and induced (dark shading).

variants display altered binding patterns in vitro. To directly examine the DNA binding of the variants, a DNA fragment containing a single iteron from pRK1 (11) was combined with purified, histidine-tagged variants over a range of protein concentrations. The preparation of the DNA fragment and protein purification were done essentially as previously described (14, 23), except that the latter used Ni-Sepharose 6 Fast Flow resin (GE Healthcare) in the place of Ni-nitrilotriacetic acid agarose (QIAGEN). Samples (15-µl final volume) for binding reactions were prepared in 1x binding buffer (2 mM Tris-HCl [pH 7.5], 0.6 mM MgCl₂, 0.1 mM EDTA, and 10 mM potassium glutamate) with 500 pg labeled DNA, 65 ng poly(dI-dC), and 1 µl diluted protein. Proteins were diluted in TGE buffer (14) to concentrations of 25, 50, 100, and 200 ng/µl. Binding reaction mixtures were incubated for 15 min at room temperature, and samples were analyzed by electrophoretic mobility shift assays (11). A representative gel is shown along with corresponding quantification data for lanes with 200 ng of each variant (Fig. 4). The results demonstrate that alanine substitutions at positions 71, 74, and 131 produced binding deficits in monomers but not dimers. For the Y74A mutant, the defect was particularly severe, suggesting that the tyrosine residue at position 74 is critical for monomer binding. Alanine substitutions at positions 225 and 254 affected the binding of both monomers and dimers. Interestingly, although the protein with a G211A substitution in the unstructured loop behaved the most like the wt in the in vivo replication assays, it showed marked reductions in both monomer and dimer binding to the DNA. We also noted that a reduction in dimer binding of 50% or more (G211A, R225A, and R254A mutants) did not weaken the inhibition of plasmid replication in the in vivo assay (Fig. 3). Importantly, however, work by Urh et al. demonstrated that DNA-binding proficiency is not required for a variant to inhibit the replication of a ori plasmid (20). To assay the ability of a variant to inhibit replication, the system must also include wt (or another active initiator form of ) to support replication. The variant does not need to have DNA-binding activity to inhibit replication initiation by the wt protein; it needs only a functional dimerization domain. Presumably, by forming heterodimers with wt , the variant protein reduces the pool of monomeric initiators.

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FIG. 4. Binding of variants to a single iteron. (A) A 3'-end-labeled DNA fragment containing a single iteron was incubated with increasing amounts (25, 50, 100, and 200 ng) of (wt or variant) and subjected to electrophoretic mobility shift assay. The positions of free DNA (F) and nucleoprotein complexes containing monomers (M) or dimers (D) are indicated. (B) Percentages of DNA bound by 200 ng of (wt or variant).

The data from the in vivo functional assays and the in vitro binding assays are consistent with roles for at least five of the six targeted amino acids in contacting the DNA. Mutations in the WH1 motif (S71A, Y74A, and G131A) disturbed monomer binding only; mutations in the WH2 domain (R225A and R254A) affected the binding of both monomers and dimers.

At the time this work was being conducted, only two Rep structures had been solved from protein crystals; the structures of other Reps, including , could only be hypothesized based on homologies in the Rep family. Recently, however, Swan et al. published the crystal structure of the protein in complex with iteron DNA (19). Consistent with our results, their data also demonstrate the importance of amino acids Arg225 and Ser71 in DNA contact and replication and support DNA contact by Tyr74. In contrast, no mention is made of Gly131. Perhaps this is because their structure was derived from a quadruple substitution variant of , or perhaps the G131A substitution indirectly disrupts DNA binding. The paper by Swan et al. presents evidence for DNA contact by two immediately preceding amino acids, Asp129 and Glu130. Additionally, it suggests the importance of Arg254, but it is not clear on what basis Arg254 is identified as contacting DNA. Here we provide data consistent with a role for Arg254 in iteron binding. Taken together, we believe the parallels between our findings bode well for the utility of homology-based structural models of Rep proteins for the dissection of structure/function relationships and for generating Rep protein variants with desired characteristics.

 
We thank Wilma Ross for help in figure preparation.

This work was supported by National Institutes of Health grant GM40314 to M.F.

 
* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin, 420 Henry Mall, Madison, WI 53706. Phone: (608) 262-6947. Fax: (608) 262-9865. E-mail: msfiluto{at}facstaff.wisc.edu

Published ahead of print on 20 April 2007.

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Journal of Bacteriology, July 2007, p. 4953-4956, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00109-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kunnimalaiyaan, S. Articles by Filutowicz, M. Search for Related Content PubMed PubMed Citation Articles by Kunnimalaiyaan, S. Articles by Filutowicz, M.