Cognate Target Sites Research Articles

High-throughput evaluation of genetic variants with prime editing sensor libraries.

Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.

Intein-based thermoregulated meganucleases for containment of genetic material.

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (TSMs) by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

Synthesis of S-Adenosyl-L-Methionine Analogs with Extended Transferable Groups for Methyltransferase-Directed Labeling of DNA and RNA.

S-Adenosyl-L-methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium-bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases, enabling covalent derivatization and subsequent labeling of their cognate target sites in DNA or RNA. Although AdoMet analogs with saturated aliphatic chains are less popular than propargylic ones, they can be useful for dedicated studies that require certain chemical derivatization. Here we describe synthetic procedures for the preparation of two AdoMet analogs, one with a transferable 6-azidohex-2-ynyl group (carrying an activating C≡C triple bond and a terminal azide functionality), and the other one with a transferable ethyl-2,2,2-d3 group (an isotope-labeled aliphatic moiety). Our synthetic approach is based on direct chemoselective alkylation of S-adenosyl-L-homocysteine at sulfur with a corresponding nosylate or triflate, respectively, under acidic conditions. We also describe synthetic routes to 6-azidohex-2-yn-1-ol and conversion of the alcohols to corresponding nosylate and triflate alkylators. Using these protocols, the synthetic AdoMet analogs can be prepared within 1 to 2 weeks. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 6-azidohex-2-yn-1-ol Basic Protocol 2: Synthesis of 4-nitrobenzenesulfonate Basic Protocol 3: Synthesis of trifluoromethanesulfonates Basic Protocol 4: S-Alkylation of AdoHcy with sulfonates Basic Protocol 5: Purification and characterization of AdoMet analogs.

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.

Base editing sensor libraries for high-throughput engineering and functional analysis of cancer-associated single nucleotide variants.

Base editing (BE) can be applied to characterize single nucleotide variants (SNVs) of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular BE-activity ‘sensors’ that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor–sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated SNVs in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models, and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput BE screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.

Dynamics of chromosomal target search by a membrane-integrated one-component receptor.

Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.

Dissecting Dynamic and Hydration Contributions to Sequence-Dependent DNA Minor Groove Recognition

Sequence selectivity is a critical attribute of DNA-binding ligands and underlines the need for detailed molecular descriptions of binding in representative sequence contexts. We investigated the binding and volumetric properties of DB1976, a model bis(benzimidazole)-selenophene diamidine compound with emerging therapeutic potential in acute myeloid leukemia, debilitating fibroses, and obesity-related liver dysfunction. To sample the scope of cognate DB1976 target sites, we evaluated three dodecameric duplexes spanning >103-fold in binding affinity. The attendant changes in partial molar volumes varied substantially, but not in step with binding affinity, suggesting distinct modes of interactions in these complexes. Specifically, whereas optimal binding was associated with loss of hydration water, low-affinity binding released more hydration water. Explicit-atom molecular dynamics simulations showed that minor groove binding perturbed the conformational dynamics and hydration at the termini and interior of the DNA in a sequence-dependent manner. The impact of these distinct local dynamics on hydration was experimentally validated by domain-specific interrogation of hydration with salt, which probed the charged axial surfaces of oligomeric DNA preferentially over the uncharged termini. Minor groove recognition by DB1976, therefore, generates dynamically distinct domains that can make favorable contributions to hydration release in both high- and low-affinity binding. Because ligand binding at internal sites of DNA oligomers modulates dynamics at the termini, the results suggest both short- and long-range dynamic effects along the DNA target that can influence their effectiveness as low-MW competitors of protein binding.

Base-pairing interactions between substrate RNA and H/ACA guide RNA modulate the kinetics of pseudouridylation, but not the affinity of substrate binding by H/ACA small nucleolar ribonucleoproteins.

H/ACA small nucleolar ribonucleoproteins (snoRNPs) pseudouridylate RNA in eukaryotes and archaea. They target many RNAs site-specifically through base-pairing interactions between H/ACA guide and substrate RNA. Besides ribosomal RNA (rRNA) and small nuclear RNA (snRNA), H/ACA snoRNPs are thought to also modify messenger RNA (mRNA) with potential impacts on gene expression. However, the base pairing between known target RNAs and H/ACA guide RNAs varies widely in nature, and therefore the rules governing substrate RNA selection are still not fully understood. To provide quantitative insight into substrate RNA recognition, we systematically altered the sequence of a substrate RNA target by the Saccharomyces cerevisiae H/ACA guide RNA snR34. Time courses measuring pseudouridine formation revealed a gradual decrease in the initial velocity of pseudouridylation upon reducing the number of base pairs between substrate and guide RNA. Changing or inserting nucleotides close to the target uridine severely impairs pseudouridine formation. Interestingly, filter binding experiments show that all substrate RNA variants bind to H/ACA snoRNPs with nanomolar affinity. Next, we showed that binding of inactive, near-cognate RNAs to H/ACA snoRNPs does not inhibit their activity for cognate RNAs, presumably because near-cognate RNAs dissociate rapidly. We discuss that the modulation of initial velocities by the base-pairing strength might affect the order and efficiency of pseudouridylation in rRNA during ribosome biogenesis. Moreover, the binding of H/ACA snoRNPs to near-cognate RNAs may be a mechanism to search for cognate target sites. Together, our data provide critical information to aid in the prediction of productive H/ACA guide—substrate RNA pairs.

Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.

Three new active members of the I-OnuI family of homing endonucleases.

In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity.

ABSTRACTAnalysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.

Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.

Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification.

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid “linker” and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the “spacer” separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5–7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

BackgroundPhosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites) has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites.ResultsWe characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D) structural information available in the protein data bank (PDB) and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information.ConclusionWhile local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as relevant for the recognition of kinases and their cognate target sites and can be used for an improved prediction of phosphorylation sites. A web-based service (Phos3D) implementing the developed structure-based P-site prediction method has been made available at .

Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease

Homing endonucleases stimulate gene conversion by generating site-specific DNA double-strand breaks that are repaired by homologous recombination. These enzymes are potentially valuable tools for targeted gene correction and genome engineering. We have engineered a variant of the I-AniI homing endonuclease that nicks its cognate target site. This variant contains a mutation of a basic residue essential for proton transfer and solvent activation in one active site. The cleavage mechanism, DNA-binding affinity, and substrate specificity profile of the nickase are similar to the wild-type enzyme. I-AniI nickase stimulates targeted gene correction in human cells, in cis and in trans, at approximately 1/4 the efficiency of the wild-type enzyme. The development of sequence-specific nicking enzymes like the I-AniI nickase will facilitate comparative analyses of DNA repair and mutagenesis induced by single- or double-strand breaks.

Prediction of TF target sites based on atomistic models of protein-DNA complexes

BackgroundThe specific recognition of genomic cis-regulatory elements by transcription factors (TFs) plays an essential role in the regulation of coordinated gene expression. Studying the mechanisms determining binding specificity in protein-DNA interactions is thus an important goal. Most current approaches for modeling TF specific recognition rely on the knowledge of large sets of cognate target sites and consider only the information contained in their primary sequence.ResultsHere we describe a structure-based methodology for predicting sequence motifs starting from the coordinates of a TF-DNA complex. Our algorithm combines information regarding the direct and indirect readout of DNA into an atomistic statistical model, which is used to estimate the interaction potential. We first measure the ability of our method to correctly estimate the binding specificities of eight prokaryotic and eukaryotic TFs that belong to different structural superfamilies. Secondly, the method is applied to two homology models, finding that sampling of interface side-chain rotamers remarkably improves the results. Thirdly, the algorithm is compared with a reference structural method based on contact counts, obtaining comparable predictions for the experimental complexes and more accurate sequence motifs for the homology models.ConclusionOur results demonstrate that atomic-detail structural information can be feasibly used to predict TF binding sites. The computational method presented here is universal and might be applied to other systems involving protein-DNA recognition.

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.

IκB Kinase ϵ Interacts with p52 and Promotes Transactivation via p65

IκB Kinase ϵ Interacts with p52 and Promotes Transactivation via p65

Isolation and characterization of new homing endonuclease specificities at individual target site positions.

Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity.

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

By stopped-flow kinetics using 2-aminopurine as a probe to detect base flipping, we show here that base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase (MTase) is a biphasic process: target base flipping is very fast (kflip>240 s−1), but binding of the flipped base into the active site pocket of the enzyme is slow (k=0.1–2 s−1). Whereas base flipping occurs in the absence of S-adenosyl-l-methionine (AdoMet), binding of the target base in the active site pocket requires AdoMet. Our data suggest that the tyrosine residue in the DPPY motif conserved in the active site of DNA-(adenine-N6)-MTases stacks to the flipped target base. Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base. The exchange of Ser188 located in a loop next to the active center by alanine led to a seven- to eightfold reduction of kflip, which was also reduced with substrates having altered GATC recognition sites and in the absence of AdoMet. These findings provide evidence that the enzyme actively initiates base flipping by stabilizing the transition state of the process. Reduced rates of base flipping in substrates containing the target base in a non-canonical sequence demonstrate that DNA recognition by the MTase starts before base flipping. DNA recognition, cofactor binding and base flipping are correlated and efficient base flipping takes place only if the enzyme has bound to a cognate target site and AdoMet is available.