From Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions

DNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Cα deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.     

A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide

Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA–protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ∼18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA–protein interactions.     

Analytical biochemistry of DNA–protein assemblies from crude cell extracts

Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.     

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.     

Dissecting protein-induced DNA looping dynamics in real time

Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA–protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA–protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA–protein dynamics in real time.     

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.     

Structure and function of the regulatory C-terminal HRDC domain from Deinococcus radiodurans RecQ

RecQ helicases are critical for maintaining genome integrity in organisms ranging from bacteria to humans by participating in a complex network of DNA metabolic pathways. Their diverse cellular functions require specialization and coordination of multiple protein domains that integrate catalytic functions with DNA–protein and protein–protein interactions. The RecQ helicase from Deinococcus radiodurans (DrRecQ) is unusual among RecQ family members in that it has evolved to utilize three ‘Helicase and RNaseD C-terminal’ (HRDC) domains to regulate its activity. In this report, we describe the high-resolution structure of the C-terminal-most HRDC domain of DrRecQ. The structure reveals unusual electrostatic surface features that distinguish it from other HRDC domains. Mutation of individual residues in these regions affects the DNA binding affinity of DrRecQ and its ability to unwind a partial duplex DNA substrate. Taken together, the results suggest the unusual electrostatic surface features of the DrRecQ HRDC domain may be important for inter-domain interactions that regulate structure-specific DNA binding and help direct DrRecQ to specific recombination/repair sites.     

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.     

Trapping DNA-protein binding reactions with neutral osmolytes for the analysis by gel mobility shift and self-cleavage assays

We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA–protein complex dissociation to develop a method that uses osmotic stress to ‘freeze’ mixtures of DNA–protein complexes and prevent further reaction enabling analysis of the products. We apply this approach to the gel mobility shift assay and use it to modify a self-cleavage assay that uses the nuclease activity of the restriction endonucleases to measure sensitively their specific binding to DNA. At sufficiently high concentrations of neutral osmolytes the cleavage reaction can be triggered at only those DNA fragments with initially bound enzyme. The self-cleavage assay allows measurement of binding equilibrium and kinetics directly in solution avoiding the intrinsic problems of gel mobility shift and filter binding assays while providing the same sensitivity level. Here we compare the self-cleavage and gel mobility shift assays applied to the DNA binding of EcoRI and BamHI restriction endonucleases. Initial results indicate that BamHI dissociation from its specific DNA sequence is strongly linked to water activity with the half-life time of the specific complex increasing ~20-fold from 0 to 1 osmolal betaine.     

Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis.     

Interaction of bacteriophage T7 gene 4 primase with its template recognition site

The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.     

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Selective Glutaraldehyde-Mediated Coupling of Proteins to the 3′-Adenine Terminus of Polymerase Chain Reaction Products

Attachment of proteins to the 3′ end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ′ modifications by solid-phase synthesis for protein attachment is limited to 45–60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3′-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N⁶ position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4°C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and β2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4°C showed no evidence of DNA–DNA or DNA–protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA–3′-protein conjugates might be easily mass-produced. The protein portion of a DNA–3′-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization.     

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3′-mismatched RNA primer because it cannot remove the 3′-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3′-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3′–5′ direction, and the kinetic parameters (K_m and K_cat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3′-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3′-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3′-mismatched RNA primer after the 3′-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3′-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.