Noncognate Sites Research Articles

A Conformational Switch in the Zinc Finger Protein Kaiso Mediates Differential Readout of Specific and Methylated DNA Sequences.

Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to the different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against noncognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.

The Role of Noncognate Sites in the 1D Search Mechanism of EcoRI

A one-dimensional (1D) search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of 1D diffusion can help resolve the competing demands of a fast search and high target affinity, a conflict known as the speed-selectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of a 1D search by quantum dot-labeled EcoRI. Our data supports the view that proteins search DNA via rotation-coupled sliding over a corrugated energy landscape. We observed that whereas EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations, its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites, which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence-independent energy landscape during fast search but rapidly interconverts with a “hemispecific” binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding, which can then lead to target recognition.

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher.

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity

Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast.

Single molecule study of non-specific binding kinetics of LacI in mammalian cells.

Many key cellular processes are controlled by the association of DNA-binding proteins (DBPs) to specific sites. The kinetics of the search process leading to the binding of DBPs to their target locus are largely determined by transient interactions with non-cognate DNA. Using single-molecule microscopy, we studied the dynamics and non-specific binding to DNA of the Lac repressor (LacI) in the environment of mammalian nuclei. We measured the distribution of the LacI-DNA binding times at non-cognate sites and determined the mean residence time to be τ(1D) = 182 ms. This non-specific interaction time, measured in the context of an exogenous system such as that of human U2OS cells, is remarkably different compared to that reported for the LacI in its native environment in E. coli (<5 ms). Such a striking difference (more than 30 fold) suggests that the genome, its organization, and the nuclear environment of mammalian cells play important roles on the dynamics of DBPs and their non-specific DNA interactions. Furthermore, we found that the distribution of off-target binding times follows a power law, similar to what was reported for TetR in U2OS cells. We argue that a possible molecular origin of such a power law distribution of residence times is the large variability of non-cognate sequences found in the mammalian nucleus by the diffusing DBPs.

Functional Implications of Intrinsic Helicities within Basic Regions of bZIP Transcription Factors

Eukaryotic transcription factors (TFs) including basic leucine zippers (bZIPs) are enriched in disorder promoting residues. In contrast to the presumption that monomeric unbound bZIP basic regions (bRs) are uniformly disordered, recent studies have shown that unbound monomeric bRs have quantifiable -helicity. The 8-residue segments, directly N-terminal to DNA-binding motifs are primary modulators of intrinsic helicities. It is conceivable that observed intrinsic helicities and their variation with bR sequences are inconsequential given that sequences of disordered regions change rapidly. Our sequence analysis of bZIPs, however, demonstrates that bRs show a high degree of conservation across orthologs and considerable variation among paralogs. Based on this, we predict that bRs from similar sequence families and different organisms are likely to have equivalent intrinsic helicities and propose that this reflects a mechanistic conservation across orthologs with implications for search process for cognate DNA half sites. To test our predictions we have performed lattice-based kinetic Monte Carlo simulations to quantify the impact of variations in intrinsic helicities on the process of DNA binding. DNA is modeled as a wormlike chain and a randomly chosen site is designated as the cognate site. We model the interplay of three-dimensional diffusion of free TF molecules in solution, binding and unbinding of TF to the non-cognate sites, and one-dimensional sliding along the DNA. The effects of intrinsic helicity on these processes are investigated through systematic titrations in a multidimensional parameter space and quantifying their effects on the first passage time distribution for TF to find its cognate site. The search time has a non-monotonic dependence on bR helicity such that the search process is optimized within a limited regime of bR helicity. We also quantify effects of bZIP dimerization as a secondary modulator of TF-DNA interactions.

Sequence-dependent sliding kinetics of p53

Proper timing of gene expression requires that transcription factors (TFs) efficiently locate and bind their target sites within a genome. Theoretical studies have long proposed that one-dimensional sliding along DNA while simultaneously reading its sequence can accelerate TF's location of target sites. Sliding by prokaryotic and eukaryotic TFs were subsequently observed. More recent theoretical investigations have argued that simultaneous reading and sliding is not possible for TFs without their possessing at least two DNA-binding modes. The tumor suppressor p53 has been shown to slide on DNA, and recent experiments have offered structural and single molecule support for a two-mode model for the protein. If the model is applicable to p53, then the requirement that TFs be able to read while sliding implies that noncognate sites will affect p53's mobility on DNA, which will thus be generally sequence-dependent. Here, we confirm this prediction with single-molecule microscopy measurements of p53's local diffusivity on noncognate DNA. We show how a two-mode model accurately predicts the variation in local diffusivity, while a single-mode model does not. We further determine that the best model of sequence-specific binding energy includes terms for "hemi-specific" binding, with one dimer of tetrameric p53 binding specifically to a half-site and the other binding nonspecifically to noncognate DNA. Our work provides evidence that the recognition by p53 of its targets and the timing thereof can depend on its noncognate binding properties and its ability to change between multiple modes of binding, in addition to the much better-studied effects of cognate-site binding.

The role of protein sequence and global conformation in DNA binding specificity of members of the Cro family

The Cro family of bacteriophage DNA‐binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are α‐helical and mixed α‐helical/β‐sheet, respectively, despite sharing 40% sequence identity. Both proteins are transcription factors that use a helix‐turn‐helix (HTH) motif as their primary DNA recognition moiety. However, the natural consensus DNA sequences of the proteins are different at three positions in each seven base‐pair half site. Fluorescence anisotropy measurements showed that wild‐type (WT) Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (KD) of 230 nM and 56 nM, respectively. WT Pfl 6 bound its noncognate site with KD = 1.99 μM and WT Xfaso 1 did not bind its noncognate site at all. To explore whether this change in binding specificity is due to differences in sequence in the HTH region or to global differences in their tertiary structures, mutations were introduced into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. The binding affinities of these variant proteins against the consensus binding sites were tested and preliminary results suggest that mutation of the Xfaso 1 sequence toward Pfl 6 allows it to bind the Pfl 6 consensus site. Funded by NSF (MCB‐0647390) and HHMI (52005889).

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved "two-handed" pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

Protein–DNA Recognition Triggered by a DNA Conformational Switch

How DNA-binding proteins find their target sites remains a fascinating question. Early work on the Lac repressor showed that proteins specifically bind faster than simple diffusion allows. This led to the idea that recognition could be accelerated by combining diffusion with sliding along DNA and hopping between neighboring strands. This, in turn, implied that proteins could interact with DNA in a distinct nonspecific manner. Experimental work has confirmed that proteins can indeed slide along DNA, although typical sliding distances vary from protein to protein. Recent work also indicates that sliding follows the helical grooves of DNA. Crystallography of proteins bound to noncognate sites, NMR spectroscopy, and molecular simulations have all provided data on nonspecific binding, notably suggesting that proteins maintain similar orientations with respect to DNA in nonspecifically and specifically bound states (see, e.g., Refs. [7–9]). However, little is known about the transition between these states, although theoretical studies have suggested that a switching mechanism may exist, possibly involving a protein conformational change. To analyze this problem at the atomic level, we carried out a molecular dynamics (MD) study on the dissociation of a specific protein–DNA complex in water, starting from the bound conformation. We studied the sex-determining region Y (SRY) protein, which affects the gender selection in mammals and is linked to a number of gender-related pathologies. The SRY protein binds in the minor groove of DNA, optimally at an (A/T)AACAAT sequence, and opens the minor groove by partial intercalation of an isoleucine residue (Ile13) between two adjacent AT base pairs and induces local unwinding and bending of DNA away from the protein. Using a specially designed restraint for the minimal atomic distance between any pair of nonhydrogen atoms across the protein–DNA interface (dMIN), we were able to control the dissociation of the SRY protein from a 14-base-pair (bp) DNA oligomer (5’-CCTGCACAAACACC-3’) without biasing the conformational pathway. Using this approach, roughly 0.6 ms of umbrella sampling led to a free-energy profile for the dissociation/association process. This profile showed a free-energy gain of 11.5 kcal mol 1 because of the SRY-DNA binding; this binding process includes passage of an energy barrier of roughly 4 kcalmol 1 at a separation of 4.2–3.5 and a secondary barrier of 2 kcalmol 1 at 3.1 (see Figure S1 in the Supporting Information). We investigated the conformational aspects of this pathway to understand the recognition mechanism. An initial analysis showed that the conformation of the SRY protein remained remarkably stable during its separation from the DNA. Although the Nand C-terminal tails were very flexible, the three-a-helix protein core varied by a root-mean-square deviation (RMSD) of 2.2 at most (see Figure S1 in the Supporting Information). In contrast, the DNA oligomer underwent considerable change, linked to the SRY-induced deformations. However, our initial analysis of the separation profile also showed that many DNA conformations and protein locations occurred for a single minimal pair distance along the separation pathway. We reduced this problem by using the distance dAXC from the center of the DNA helical axis to the center of mass of the core region of the SRY protein. This distance varies almost monotonically with the minimal pair distance for dMIN 20 . The most interesting feature lies between these two regions (13 > dAXC> 20 ), where the DNA conformations clearly split into two paths (green and red). The center of the two-path region occurs around dAXC= 16 . Here, both paths are found at a RMSD value of around 3 relative to the bound DNA reference state, whereas the upper path (path 1) is located at a RMSD value of 5.2 relative to the unbound [*] Dr. B. Bouvier, Dr. K. Zakrzewska, Dr. R. Lavery Universit Lyon 1/CNRS, UMR 5086, Bases Mol culaires et Structurales des Syst mes Infectieux, IBCP 7 passage du Vercors, 69367 Lyon (France) E-mail: richard.lavery@ibcp.fr Homepage: http://www.ibcp.fr

Fidelity of DNA Sequence Recognition by the SfiI Restriction Endonuclease Is Determined by Communications between Its Two DNA-Binding Sites

The SfiI restriction endonuclease is a tetramer in which two subunits form a dimeric unit that contains one DNA binding cleft and the other two subunits contain a second cleft on the opposite side of the protein. Full activity requires both clefts to be filled with its recognition sequence: SfiI has low activity when bound to one site. The ability of SfiI to cleave non-cognate sites, one base pair different from the true site, was initially tested on substrates that lacked specific sites but which contained either one or multiple non-cognate sites. No cleavage of the DNA with one non-cognate site was detected, while a small fraction of the DNA with multiple sites was nicked. The alternative sequences were, however, cleaved in both strands, albeit at low levels, when the DNA also carried either a recognition site for SfiI or the termini generated by SfiI. Further tests employed a mutant of SfiI, altered at the dimer interface, which was known to be more active than wild-type SfiI when bound to a single site. This mutant similarly failed to cleave DNA with one non-cognate site, but cleaved the substrates with multiple non-cognate sites more readily than did the native enzyme. To cleave additional sites, SfiI thus needs to interact concurrently with either two non-cognate sites or one non-cognate and one cognate site (or the termini thereof), yet this arrangement is still restrained from cleaving the alternative site unless the communication pathway between the two DNA-binding clefts is disrupted.

Two Yeast PUF Proteins Negatively Regulate a Single mRNA

mRNA stability and translation are regulated by protein repressors that bind 3'-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3'-untranslated region-mediated control.

The GCN4 bZIP Targets Noncognate Gene Regulatory Sequences: Quantitative Investigation of Binding at Full and Half Sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.

Determinants of Sequence-Specific DNA Methylation: Target Recognition and Catalysis Are Coupled in M.HhaI

Sequence specificity studies of the wild-type bacterial DNA cytosine C5 methyltransferase HhaI were carried out with cognate (5'GCGC3') and noncognate DNA substrates containing single base pair changes at the first and the fourth position (underlined). Specificity for noncognate site methylation at the level of kcat/KDDNA is decreased 9000-80000-fold relative to the cognate site, manifested through changes in methylation, or a prior step, and changes in KDDNA. Analysis of a new high-resolution enzyme-DNA cocrystal structure provides a partial mechanistic understanding of this discrimination. To probe the significance of conformational transitions occurring prior to catalysis in determining specificity, we analyzed the double mutant (H127A/T132A). These amino acid substitutions disrupt the interface between the flexible loop (residues 80-99), which interacts with the DNA minor groove, and the active site. The mutant's methylation of the cognate site is essentially unchanged, yet its methylation of noncognate sites is decreased up to 460-fold relative to the wild-type enzyme. We suggest that a significant contribution to M.HhaI's specificity involves the stabilization of reaction intermediates prior to methyl transfer, mediated by DNA minor groove-protein flexible loop interactions.

Conformational Transitions as Determinants of Specificity for the DNA Methyltransferase EcoRI

Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500- and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an approximately 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.

Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, “flipped” cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl- l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme–DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.

Detecting transient intermediates in macromolecular binding by paramagnetic NMR

Macromolecular complex formation is governed by two opposing constraints of specificity and speed. Kinetic and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process or by the creation of a short-range attractive potential around the target site. This implies the existence of transient intermediates involving non-specific binding modes. Here we show that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence of, and investigating the nature of, low population transient intermediates under equilibrium conditions. Applying this approach, we characterize the search process whereby a sequence-specific transcription factor (the homeodomain of HOXD9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. The intermolecular PRE method is general and can be readily applied to investigations of transient intermediates in many other macromolecular binding processes.

Non-cognate Enzyme–DNA Complex: Structural and Kinetic Analysis of EcoRV Endonuclease Bound to the EcoRI Recognition Site GAATTC

The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.

Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase

DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.

Catalytic efficiency and sequence selectivity of a restriction endonuclease modulated by a distal manganese ion binding site

Crystal structures of EcoRV endonuclease bound in a ternary complex with cognate duplex DNA and manganese ions have previously revealed an Mn 2+-binding site located between the enzyme and the DNA outside of the dyad-symmetric GATATC recognition sequence. In each of the two enzyme subunits, this metal ion bridges between a distal phosphate group of the DNA and the imidazole ring of His71. The new metal-binding site is specific to Mn 2+ and is not occupied in ternary cocrystal structures with either Mg 2+ or Ca 2+. Characterization of the H71A and H71Q mutants of EcoRV now demonstrates that these distal Mn 2+ sites significantly modulate activity toward both cognate and non-cognate DNA substrates. Single-turnover and steady-state kinetic analyses show that removal of the distal site in the mutant enzymes increases Mn 2+-dependent cleavage rates of specific substrates by tenfold. Conversely, the enhancement of non-cognate cleavage at GTTATC sequences by Mn 2+ is significantly attenuated in the mutants. As a consequence, under Mn 2+ conditions EcoRV-H71A and EcoRV-H71Q are 100 to 700-fold more specific than the wild-type enzyme for cognate DNA relative to the GTTATC non-cognate site. These data reveal a strong dependence of DNA cleavage efficiency upon metal ion-mediated interactions located some 20 Å distant from the scissile phosphodiester linkages. They also show that discrimination of cognate versus non-cognate DNA sequences by EcoRV depends in part on contacts with the sugar-phosphate backbone outside of the target site.