Recognition Of Single-stranded DNA Research Articles

R-loop-dependent promoter-proximal termination ensures genome stability

The proper regulation of transcription is essential for maintaining genome integrity and executing other downstream cellular functions1,2. Here we identify a stable association between the genome-stability regulator sensor of single-stranded DNA (SOSS)3 and the transcription regulator Integrator-PP2A (INTAC)4–6. Through SSB1-mediated recognition of single-stranded DNA, SOSS–INTAC stimulates promoter-proximal termination of transcription and attenuates R-loops associated with paused RNA polymerase II to prevent R-loop-induced genome instability. SOSS–INTAC-dependent attenuation of R-loops is enhanced by the ability of SSB1 to form liquid-like condensates. Deletion of NABP2 (encoding SSB1) or introduction of cancer-associated mutations into its intrinsically disordered region leads to a pervasive accumulation of R-loops, highlighting a genome surveillance function of SOSS–INTAC that enables timely termination of transcription at promoters to constrain R-loop accumulation and ensure genome stability.

Structure, stability and specificity of the binding of ssDNA and ssRNA with proteins.

Recognition of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) is important for many fundamental cellular functions. A variety of single-stranded DNA-binding proteins (ssDBPs) and single-stranded RNA-binding proteins (ssRBPs) have evolved that bind ssDNA and ssRNA, respectively, with varying degree of affinities and specificities to form complexes. Structural studies of these complexes provide key insights into their recognition mechanism. However, computational modeling of the specific recognition process and to predict the structure of the complex is challenging, primarily due to the heterogeneity of their binding energy landscape and the greater flexibility of ssDNA or ssRNA compared with double-stranded nucleic acids. Consequently, considerably fewer computational studies have explored interactions between proteins and single-stranded nucleic acids compared with protein interactions with double-stranded nucleic acids. Here, we report a newly developed energy-based coarse-grained model to predict the structure of ssDNA–ssDBP and ssRNA–ssRBP complexes and to assess their sequence-specific interactions and stabilities. We tuned two factors that can modulate specific recognition: base–aromatic stacking strength and the flexibility of the single-stranded nucleic acid. The model was successfully applied to predict the binding conformations of 12 distinct ssDBP and ssRBP structures with their cognate ssDNA and ssRNA partners having various sequences. Estimated binding energies agreed well with the corresponding experimental binding affinities. Bound conformations from the simulation showed a funnel-shaped binding energy distribution where the native-like conformations corresponded to the energy minima. The various ssDNA–protein and ssRNA–protein complexes differed in the balance of electrostatic and aromatic energies. The lower affinity of the ssRNA–ssRBP complexes compared with the ssDNA–ssDBP complexes stems from lower flexibility of ssRNA compared to ssDNA, which results in higher rate constants for the dissociation of the complex (koff) for complexes involving the former.

Turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA based upon Y-shaped DNA structure

A novel turn-off colorimetric sensor for sequence-specific recognition of single-stranded DNA (ssDNA) was established by combining Y-shaped DNA duplex and G-quadruplex-hemin DNAzyme. A G-rich single-stranded DNA (Oligo-1) displays peroxidase mimicking catalytic activity due to the specific binding with hemin in the presence of K+, which was able to catalyze the oxidation of colorless 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS2−) by H2O2 to generate green ABTS•− radical for colorimetric assay. Oligonucleotide 2 (Oligo-2) was partly complementary with Oligo-1 and the target DNA. Upon addition of target DNA, Oligo-1, Oligo-2 and target DNA can hybridize with each other to form Y-shaped DNA duplex. The DNAzyme sequence of Oligo-1 was partly caged into Y-shaped DNA duplex, resulting in the inactivation of the DNAzyme and a sharp decrease of the absorbance of the oxidation product of ABTS2−. Under the optimum condition, the absorbance decreased linearly with the concentration of target DNA over the range of 1.0–250 nM and the detection limit was 0.95 nM (3σ/slope) Moreover, satisfied result was obtained for the discrimination of single-base or two-base mismatched DNA.

A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA.

A fluorescent method is described for simultaneous recognition of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). It is based on the quenching of the fluorescence of fluorophore labeled DNA probes by gold nanoparticles (AuNPs). To demonstrate feasibility, two DNA probes labeled with spectrally different fluorophores were designed. The first DNA probe (P1) was modified with 6-carboxyfluorescein (FAM; with green fluorescence, peaking at 518nm), while the second (P2) was modified with carboxy-X-rhodamine (ROX; with yellow fluorescence, 610nm). The fluorescence signals of the labels are quenched if P1 or P2 are adsorbed on AuNPs. Upon addition of ssDNA and dsDNA, hybridization occurs between P1 and ssDNA to form a dsDNA. In contrast, P2 hybridizes with dsDNA such that a triplex DNA is formed. As a result, the dsDNA and the triplex DNA, respectively, are desorbed from the surface of the AuNPs so that quenching no longer can occur and strong fluorescence can be observed. Under the optimal conditions, ssDNA and dsDNA can be detected simultaneously via the green and yellow fluorescence, respectively. The detection limits can be as low as 330 pM. In particular, the method has excellent selectivity for the target DNAs over control DNAs. Graphical abstract A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA is developed based on the fluorescence quenching of gold nanoparticles to different fluorophore labeled DNA probes.

Tying up the Ends: Plasticity in the Recognition of Single-Stranded DNA at Telomeres.

Telomeres terminate nearly exclusively in single-stranded DNA (ssDNA) overhangs comprised of the G-rich 3' end. This overhang varies widely in length from species to species, ranging from just a few bases to several hundred nucleotides. These overhangs are not merely a remnant of DNA replication but rather are the result of complex further processing. Proper management of the telomeric overhang is required both to deter the action of the DNA damage machinery and to present the ends properly to the replicative enzyme telomerase. This Current Topic addresses the biochemical and structural features used by the proteins that manage these variable telomeric overhangs. The Pot1 protein tightly binds the single-stranded overhang, preventing DNA damage sensors from binding. Pot1 also orchestrates the access of telomerase to that same substrate. The remarkable plasticity of the binding interface exhibited by the Schizosaccharomyces pombe Pot1 provides mechanistic insight into how these roles may be accomplished, and disease-associated mutations clustered around the DNA-binding interface in the hPOT1 highlight the importance of this function. The budding yeast Cdc13-Stn1-Ten1, a telomeric RPA complex closely associated with telomere function, also interacts with ssDNA in a fashion that allows degenerate sequences to be recognized. A related human complex composed of hCTC1, hSTN1, and hTEN1 has recently emerged with links to both telomere maintenance and general DNA replication and also exhibits mutations associated with telomere pathologies. Overall, these sequence-specific ssDNA binders exhibit a range of recognition properties that allow them to perform their unique biological functions.

Electrostatic and Hydrophobic Interactions Mediate Single-Stranded DNA Recognition and Acta2 Repression by Purine-Rich Element-Binding Protein B.

Myofibroblast differentiation is characterized by an increased level of expression of cytoskeletal smooth muscle α-actin. In human and murine fibroblasts, the gene encoding smooth muscle α-actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5' promoter-enhancer in response to environmental cues signaling tissue repair and remodeling. Purine-rich element-binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5' polypurine sequence containing an inverted MCAT cis element required for gene activation. In this study, we evaluated the chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine-rich strand of the MCAT element in the mouse Acta2 promoter. Quantitative single-stranded DNA (ssDNA) binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent suggested that the assembly of a high-affinity nucleoprotein complex is driven by a combination of electrostatic and hydrophobic interactions. Consistent with the results of pH titration analysis, site-directed mutagenesis revealed several basic amino acid residues in the intermolecular (R267) and intramolecular (K82 and R159) subdomains that are essential for Purβ transcriptional repressor function in Acta2 promoter-reporter assays. In keeping with their diminished Acta2 repressor activity in fibroblasts, purified Purβ variants containing an R267A mutation exhibited reduced binding affinity for purine-rich ssDNA. Moreover, certain double and triple-point mutants were also defective in binding to the Acta2 corepressor protein, Y-box-binding protein 1. Collectively, these findings establish the repertoire of noncovalent interactions that account for the unique structural and functional properties of Purβ.

Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition

Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ~30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition.

Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains

During conjugative plasmid transfer, one plasmid strand is cleaved and transported to the recipient bacterium. For F and related plasmids, TraI contains the relaxase or nickase activity that cleaves the plasmid DNA strand. F TraI36, the F TraI relaxase domain, binds a single-stranded origin of transfer (oriT) DNA sequence with high affinity and sequence specificity. The TraI36 domain from plasmid R100 shares 91% amino acid sequence identity with F TraI36, but its oriT DNA binding site differs by two of eleven bases. Both proteins readily distinguish between F and R100 binding sites. In earlier work, two amino acid substitutions in the DNA binding cleft were shown to be sufficient to change the R100 TraI36 DNA-binding specificity to that of F TraI36. In contrast, three substitutions could make F TraI36 more “R100-like”, but failed to completely alter the specificity. Here we identify one additional amino acid substitution that completes the specificity swap from F to R100. To our surprise, adding further substitutions from R100 to the F background were detrimental to binding instead of being neutral, indicating that their effects were influenced by their structural context. These results underscore the complex and subtle nature of DNA recognition by relaxases and have implications for the evolution of relaxase binding sites and oriT sequences.

Combinatorial recognition of a complex telomere repeat sequence by the Candida parapsilosis Cdc13AB heterodimer.

The telomere repeat units of Candida species are substantially longer and more complex than those in other organisms, raising interesting questions concerning the recognition mechanisms of telomere-binding proteins. Herein we characterized the properties of Candida parapsilosis Cdc13A and Cdc13B, two paralogs that are responsible for binding and protecting the telomere G-strand tails. We found that Cdc13A and Cdc13B can each form complexes with itself and a heterodimeric complex with each other. However, only the heterodimer exhibits high-affinity and sequence-specific binding to the telomere G-tail. EMSA and crosslinking analysis revealed a combinatorial mechanism of DNA recognition, which entails the A and B subunit making contacts to the 3′ and 5′ region of the repeat unit. While both the DBD and OB4 domain of Cdc13A can bind to the equivalent domain in Cdc13B, only the OB4 complex behaves as a stable heterodimer. The unstable Cdc13ABDBD complex binds G-strand with greatly reduced affinity but the same sequence specificity. Thus the OB4 domains evidently contribute to binding by promoting dimerization of the DBDs. Our investigation reveals a rare example of combinatorial recognition of single-stranded DNA and offers insights into the co-evolution of telomere DNA and cognate binding proteins.

A sensitive fluorescence method for sequence-specific recognition of single-stranded DNA by using glucose oxidase

A sensitive fluorescence method was developed for sequence-specific recognition of single-stranded DNA on the surface of silver-coated glass by means of the amplification properties of glucose oxidase.

Sequence-specific recognition of single-stranded DNA using atomic absorption spectrometry

A technique for the sequence-specific recognition of single-stranded DNA using gold nanoparticles and silver-coated glass modified with oligonucleotides was developed, with determination by atomic absorption spectrometry.

Structural Basis of Multisite Single-Stranded DNA Recognition and ACTA2 Repression by Purine-Rich Element Binding Protein B (Purβ)

A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to derepression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.

Structural Basis for 5′-End-Specific Recognition of Single-Stranded DNA by the R3H Domain from Human Sμbp-2

The R3H domain is a conserved sequence motif in nucleic acid binding proteins. Previously, we reported the solution structure of the R3H domain and identified a putative nucleic acid binding site composed of three conserved basic residues [Liepinsh, E., Leonchiks, A., Sharipo, A., Guignard, L. & Otting, G. (2003). Solution structure of the R3H domain from human Sμbp-2. J. Mol. Biol.326, 217-223]. Here, we determine the binding affinities of mononucleotides and dinucleotides for the R3H domain from human Sμbp-2 (Sμbp2-R3H) and map their binding sites on the protein's surface. Although the binding affinities show up to 260-fold selectivity between different nucleotides, their binding sites and conformations seem very similar. Further, we report the NMR structure of the Sμbp2-R3H in complex with deoxyguanosine 5'-monophosphate (dGMP) mimicking the 5'-end of single-stranded DNA. Pseudocontact shifts from a paramagnetic lanthanide tag attached to residue 731 in the mutant A731C confirmed that binding of dGMP brings a loop of the protein into closer proximity. The structure provides the first structural insight into single-stranded nucleic acid recognition by the R3H domain and shows that the R3H domain specifically binds the phosphorylated 5'-end through electrostatic interactions with the two conserved arginines and stacking interactions with the highly conserved histidine.

Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

A β-sheet miniprotein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)(2), binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet miniprotein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well-structured β-hairpin made of strands 2 and 3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein redesign.

Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

Determination of Listeria Monocytogenes in Milk Samples by Signal Amplification Quartz Crystal Microbalance Sensor

A piezoelectric crystal biosensor method has been established for detection of listeria monocytogenes (LM) by gold nanoparticles (GNPs) signal amplification. Based on specific recognition of single-stranded DNA (ssDNA) hybridization, a sandwich format of ssDNA probes/complimentary ssDNA probes modified by GNPs/ssDNA of LM was fabricated. This novel strategy allows the detection limit of LM as low as 10 cfu/mL. The biosensor showed friendly biocompatibility, good specificity, acceptable stability, which provided a potential alternative tool for detection of LM in real samples. Furthermore, the developed method could be easily extended to other gene analysis schemes.

Nonadditivity in the Recognition of Single-Stranded DNA by the Schizosaccharomyces pombe Protection of Telomeres 1 DNA-Binding Domain, Pot1-DBD

The Schizosaccharomyces pombe protection of telomeres 1 (SpPot1) protein recognizes the 3' single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1-389), which can be further separated into two distinct domains, Pot1pN (residues 1-187) and Pot1pC (residues 188-389). Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a K(D) of 400 +/- 70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere.

Insights into the Dynamics of Specific Telomeric Single-Stranded DNA Recognition by Pot1pN

The N-terminal oligonucleotide/oligosaccharide-binding fold domain of the Schizosaccharomyces pombe protection of telomeres 1 (Pot1) protein, Pot1pN (residues 1-187 of full-length Pot1), specifically recognizes telomeric single-stranded DNA (ssDNA) via a complex series of molecular interactions that are punctuated by unusual internucleotide hydrogen bonds. While the structure of ssDNA-bound Pot1pN provides an initial model for understanding how the Pot1pN-ssDNA complex is assembled and how specific nucleotide recognition occurs, further refinement requires knowledge of the ssDNA-free state of Pot1pN and the dynamic changes that accompany the binding of ssDNA. Using NMR strategies, we found that ssDNA-free Pot1pN adopts a similar overall protein backbone topology as ssDNA-bound Pot1pN does. Although the backbone structure remained relatively unchanged, we observed unexpected differential dynamic changes within the ssDNA-binding pockets of Pot1pN upon binding of cognate ssDNA. These studies support a model in which conformational selection and induced fit play important roles in the recognition of ssDNA by Pot1pN. Furthermore, the studies presented here provide a more comprehensive understanding of how specific nucleotide recognition is achieved by the telomere-end protection family of essential proteins.

A Simple Cytosine to G‐Clamp Nucleobase Substitution Enables Chiral γ‐PNAs to Invade Mixed‐Sequence Double‐Helical B‐form DNA

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. [1] Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. [2] While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, [3–5] polyamides, [6–8] and zinc-finger-binding pep

Strand Invasion of Mixed-Sequence B-DNA by Acridine-Linked, γ-Peptide Nucleic Acid (γ-PNA)

Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson-Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized gamma-peptide nucleic acid (gamma-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson-Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.