Asymmetric DNA Sequence Research Articles

A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

BackgroundSomatic embryogenesis is a phenomenon carried out in an environment that generates abiotic stress. Thus, regenerants may differ from the source of explants at the morphological, genetic, and epigenetic levels. The DNA changes may be the outcome of induction media ingredients (i.e., copper and silver ions) and their concentrations and time of in vitro cultures.ResultsThis study optimised the level of copper and silver ion concentration in culture media parallel with the induction medium longevity step towards obtaining barley regenerants via somatic embryogenesis with a minimum or maximum level of tissue culture-induced differences between the donor plant and its regenerants. The optimisation process is based on tissue culture-induced variation evaluated via the metAFLP approach for regenerants derived under varying in vitro tissue culture conditions and exploited by the Taguchi method. In the optimisation and verification experiments, various copper and silver ion concentrations and the different number of days differentiated the tested trials concerning the tissue culture-induced variation level, DNA demethylation, and de novo methylation, including symmetric (CG, CHG) and asymmetric (CHH) DNA sequence contexts. Verification of optimised conditions towards obtaining regenerants with minimum and maximum variability compared to donor plants proved useful. The main changes that discriminate optimised conditions belonged to DNA demethylation events with particular stress on CHG context.ConclusionsThe combination of tissue culture-induced variation evaluated for eight experimental trials and implementation of the Taguchi method allowed the optimisation of the in vitro tissue culture conditions towards the minimum and maximum differences between a source of tissue explants (donor plant) and its regenerants from somatic embryos. The tissue culture-induced variation characteristic is mostly affected by demethylation with preferences towards CHG sequence context.

The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

BackgroundThe a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved.Methodology/Principal FindingsWe have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg2+-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3′ overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5′-GACGGGAAGACCCT-3′) and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA.Conclusions/SignificanceThe present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.

Microscopic Rearrangement of Bound Minor Groove Binders Detected by NMR

Thermodynamic and structural studies are commonly utilized to optimize small molecules for specific DNA interactions, and, thus, a significant amount of binding data is available. However, the dynamic processes that are involved in minor groove complex formation and maintenance are not fully understood. To help define the processes involved, we have conducted 1D and 2D NMR in conjunction with biosensor-SPR experiments with a variety of compounds and symmetric, as well as asymmetric, AT tract DNA sequences. Surprisingly, the NMR data clearly show exchange between equivalent binding sites for strongly binding compounds like netropsin and DB921 (Ka > 10(8) M(-1)) that does not involve dissociation off the DNA. A quantitative analysis of the data revealed that these bound exchange rates are indeed much faster than the macroscopic dissociation rates which were independently determined by biosensor-SPR. Additionally, we could show the existence of at least two 1:1 compound DNA complexes at the same site for the interaction of these compounds with an asymmetric DNA sequence. To explain this behavior we introduced a model in which the ligand is rapidly flipping between two orientations while in close association with the DNA. The ligand reorientation will contribute favorably to the binding entropy. As the potential of minor groove binders to form more than a single complex with asymmetric, as well as symmetric, duplexes is widely unknown, the consequences for binding thermodynamics and compound design are discussed.

Structure-guided reprogramming of serine recombinase DNA sequence specificity

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.

Functional analysis of MmeI from methanol utilizer Methylophilus methylotrophus, a subtype IIC restriction-modification enzyme related to type I enzymes.

MmeI from Methylophilus methylotrophus belongs to the type II restriction-modification enzymes. It recognizes an asymmetric DNA sequence, 5'-TCCRAC-3' (R indicates G or A), and cuts both strands at fixed positions downstream of the specific site. This particular feature has been exploited in transcript profiling of complex genomes (using serial analysis of gene expression technology). We have shown previously that the endonucleolytic activity of MmeI is strongly dependent on the presence of S-adenosyl-l-methionine (J. Nakonieczna, J. W. Zmijewski, B. Banecki, and A. J. Podhajska, Mol. Biotechnol. 37:127-135, 2007), which puts MmeI in subtype IIG. The same cofactor is used by MmeI as a methyl group donor for modification of an adenine in the upper strand of the recognition site to N(6)-methyladenine. Both enzymatic activities reside in a single polypeptide (919 amino acids [aa]), which puts MmeI also in subtype IIC of the restriction-modification systems. Based on a molecular model, generated with the use of bioinformatic tools and validated by site-directed mutagenesis, we were able to localize three functional domains in the structure of the MmeI enzyme: (i) the N-terminal portion containing the endonucleolytic domain with the catalytic Mg2+-binding motif D(70)-X(9)-EXK(82), characteristic for the PD-(D/E)XK superfamily of nucleases; (ii) a central portion (aa 310 to 610) containing nine sequence motifs conserved among N(6)-adenine gamma-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.

The Recognition Domain of the BpuJI Restriction Endonuclease in Complex with Cognate DNA at 1.3-Å Resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein–DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix–turn–helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.

Spatially Directed Assembly of a Heterotetrameric Cre-Lox Synapse Restricts Recombination Specificity

The pseudo-fourfold homotetrameric synapse formed by Cre protein and target DNA restricts site-specific recombination to sequences containing dyad-symmetric Cre-binding repeats. Mixtures of engineered altered-specificity Cre monomers can form heterotetramers that recombine nonidentical asymmetric sequences, allowing greater flexibility for target site selection in the genome of interest. However, the variety of tetramers allowed by random subunit association increases the chances of unintended reactivity at nontarget sites. This problem can be circumvented by specifying a unique spatial arrangement of heterotetramer subunits. By reconfiguring intersubunit protein–protein contacts, we directed the assembly of two different Cre monomers, each having a distinct DNA sequence specificity, in an alternating (ABAB) configuration. This designed heterotetramer preferentially recombined a particular pair of asymmetric Lox sites over other pairs, whereas a mixture of freely associating subunits showed little bias. Alone, the engineered monomers had reduced reactivity towards both dyad-symmetric and asymmetric sites. Specificity arose because the organization of Cre-binding repeats of the preferred substrate matched the programmed arrangement of the subunits in the heterotetrameric synapse. When this “spatial matching” principle is applied, Cre-mediated recombination can be directed to asymmetric DNA sequences with greater fidelity.

Restriction endonuclease BpuJI specific for the 5′-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.

Identification of a new subfamily of HNH nucleases and experimental characterization of a representative member, HphI restriction endonuclease

The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Cleavage of Individual DNA Strands by the Different Subunits of the Heterodimeric Restriction Endonuclease BbvCI

BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R(1), which acts at GC downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R(1)R(2) heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R(1)(-)R(2)(+) and R(1)(+)R(2)(-) mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R(1) subunit. The other strand is then cleaved slowly by R(2) before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R(1)(+) and R(2)(+) subunits in the wild-type enzyme can differ from their activities in the R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.

Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I

Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5′-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-Å resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P2 12 12, with a=164.3, b=293.0, c=71.1 Å, and contain two to four copies of the protein in the asymmetric unit.

How the BfiI restriction enzyme uses one active site to cut two DNA strands.

Unlike other restriction enzymes, BfiI functions without metal ions. It recognizes an asymmetric DNA sequence, 5'-ACTGGG-3', and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like FokI, dimerize transiently during catalysis. BfiI is also a dimer but it has only one active site, at the dimer interface. We show here that BfiI remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and/or the DNA in the BfiI-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting BfiI into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5' phosphate in the bottom strand. BfiI thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.

The Type IIs Restriction Endonuclease BspMI Is a Tetramer That Acts Concertedly at Two Copies of an Asymmetric DNA Sequence

Type IIs endonucleases recognize asymmetric DNA sequences and cleave both strands at fixed positions downstream of the sequence. Many type IIs enzymes, including BspMI, cleave substrates with two sites more rapidly than those with one site. They usually act sequentially on DNA with two sites, but BspMI converted such a substrate directly to the final products cut at both sites. The BspMI endonuclease was found to be a tetramer, in contrast to the monomeric structures for many type IIs enzymes. No change in subunit association occurred during the BspMI reaction. Plasmids with two BspMI sites were cleaved in cis, in reactions spanning sites in the same DNA, even when the sites were separated by just 38 bp. Plasmids with one BspMI site were cleaved in trans, with the enzyme bridging sites in separate DNA molecules: these slow reactions could be accelerated by adding a second DNA with the recognition sequence. Thus, whereas many type IIs enzymes dimerize before cleaving DNA, a process facilitated by two recognition sites in cis, the BspMI tetramer binds two copies of its recognition sequence before cleaving the DNA in both strands at both sites.

Many type IIs restriction endonucleases interact with two recognition sites before cleaving DNA.

Type IIs restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions, typically several base pairs away from the recognition site. These enzymes are generally monomers that transiently associate to form dimers to cleave both strands. Their reactions could involve bridging interactions between two copies of their recognition sequence. To examine this possibility, several type IIs enzymes were tested against substrates with either one or two target sites. Some of the enzymes cleaved the DNA with two target sites at the same rate as that with one site, but most cut their two-site substrate more rapidly than the one-site DNA. In some cases, the two sites were cut sequentially, at rates that were equal to each other but that exceeded the rate on the one-site DNA. In another case, the DNA with two sites was cleaved rapidly at one site, but the residual site was cleaved at a much slower rate. In a further example, the two sites were cleaved concertedly to give directly the final products cut at both sites. Many type IIs enzymes thus interact with two copies of their recognition sequence before cleaving DNA, although via several different mechanisms.

Specificity of DNA binding and methylation by the M. FokI DNA methyltransferase

The M. FokI adenine- N 6 DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine- N 6 DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M. FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2–3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C mATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein–protein interaction assays show that both domains of M. FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M. FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.

Isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric DNA sequences containing the TTAA and TTAC subsites.

A novel single-chain (sc) protein framework containing covalently dimerized DNA-binding domains (DBD) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant DBDs with altered specificities. The library members contain one wild-type DBD and one mutant domain with randomized amino acids in the DNA-contacting region. Based on previous studies, the mutant sc derivatives are expected to recognize a general ACAA-6 bp-NNNN sequence, where ACAA is contacted by the wild-type and NNNN by the mutant domain. In principle, any sequence can stand for NNNN and serve as a selection target. Here an in vivo library screening method was used to isolate mutant sc repressors that interact with an asymmetric operator containing the TTAA target. Several mutants showed high affinity in vitro binding to operators containing the target and strong (up to 80-fold) preference for the TTAA target over the wild-type TTGT. Specificity studies revealed that certain mutants bound with substantially higher affinities (K(d) approximately 10(-11)M) to operators containing the TTAC sequence, a close homolog of the TTAA target. Thus, we have fortuitously isolated mutant sc repressors that show up to a several hundred-fold preference for TTAC over TTGT.

Assemblies of replication initiator protein on symmetric and asymmetric DNA sequences depend on multiple protein oligomerization surfaces

The π 35.0 protein of plasmid R6K regulates transcription and replication by binding a DNA sequence motif (TGAGR) arranged either asymmetrically into 22 bp direct repeats (DRs) in the γ origin, or symmetrically into inverted half-repeats (IRs) in the operator of its own gene, pir. The binding patterns of the two natural forms of the π protein and their heterodimers revealed that the predominant species, π 35.0 (35.0 kDa), can bind to a single copy of the DR as either a monomer or a dimer while π 30.5 (30.5 kDa) binds only as a dimer. We demonstrate that only one subunit of a π 35.0 dimer makes specific contact with DNA. Electron microscopic (EM) analysis of the nucleoprotein complexes formed by π 35.0 and DNA fragments containing all seven DRs revealed coupled (“hand-cuffed”) DNA molecules that are aligned in a parallel orientation. Antiparallel orientations of the DNA were not observed. Thus, hand-cuffing depends on a highly ordered oligomerization of π 35.0 in such structures. The π protein (π 35.0, π 30.5) binds to an IR as a dimer or heterodimer but not as a monomer. Moreover, a single amino acid residue substitution, F200S ( pir200), introduced into π 30.5 severely destabilizes dimers of this protein in solution and concomitantly prevents binding of this protein to the IR. This mutation also changes the stability of π 35.0 dimers but it does not change the ability of π 35.0 to bind IRs. To explain these observations we propose that the diverse interactions of π variants with DNA are controlled by multiple surfaces for protein oligomerization.