Specific binding of single-stranded telomeric DNA by Cdc13p of Saccharomyces cerevisiae

Cdc13p is a single strand telomere-binding protein of Saccharomyces cerevisiae; its telomere-binding region is within amino acids 451-693, Cdc13(451-693)p. In this study, we used purified Cdc13p and Cdc13(451-693)p to characterize their telomere binding activity. We found that the binding specificity of single-stranded TG(1-3) DNA by these two proteins is similar. However, the affinity of Cdc13(451-693)p to DNA was slightly lower than that of Cdc13p. The binding of telomeric DNA by these two proteins was disrupted at NaCl concentrations higher than 0.3 m, indicating that electrostatic interaction contributed significantly to the binding process. Because both proteins bound to strand TG(1-3) DNA positioned at the 3' end, the 5' end, or in the middle of the oligonucleotide substrates, our results indicated that the location of TG(1-3) in single-stranded DNA does not appear to be important for Cdc13p binding. Moreover, using DNase I footprint analysis, the structure of the telomeric DNA complexes of Cdc13p and Cdc13(451-693)p was analyzed. The DNase I footprints of these two proteins to three different telomeric DNA substrates were virtually identical, indicating that the telomere contact region of Cdc13p is within Cdc13(451-693)p. Together, the binding properties of Cdc13p and its binding domain support the theory that the specific binding of Cdc13p to telomeres is an important feature of telomeres that regulate telomerase access and/or differentiate natural telomeres from broken ends.     

Rap1 binds single-stranded DNA at telomeric double- and single-stranded junctions and competes with Cdc13 protein

The ends of eukaryotic chromosomes are protected by specialized telomere chromatin structures. Rap1 and Cdc13 are essential for the formation of functional telomere chromatin in budding yeast by binding to the double-stranded part and the single-stranded 3' overhang, respectively. We analyzed the binding properties of Saccharomyces castellii Rap1 and Cdc13 to partially single-stranded oligonucleotides, mimicking the junction of the double- and single-stranded DNA (ds-ss junction) at telomeres. We determined the optimal and the minimal DNA setup for a simultaneous binding of Rap1 and Cdc13 at the ds-ss junction. Remarkably, Rap1 is able to bind to a partially single-stranded binding site spanning the ds-ss junction. The binding over the ds-ss junction is anchored in a single double-stranded hemi-site and is stabilized by a sequence-independent interaction of Rap1 with the single-stranded 3' overhang. Thus, Rap1 is able to switch between a sequence-specific and a nonspecific binding mode of one hemi-site. At a ds-ss junction configuration where the two binding sites partially overlap, Rap1 and Cdc13 are competing for the binding. These results shed light on the end protection mechanisms and suggest that Rap1 and Cdc13 act together to ensure the protection of both the 3' and the 5' DNA ends at telomeres.     

Identification of the determinants for the specific recognition of single-strand telomeric DNA by Cdc13

The single-strand overhang present at telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in Saccharomyces cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoan Oxytricha nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13 DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only three of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.     

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.     

Structural basis for DNA recognition and processing by UvrB

DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.     

Tetraplex formation of surface-immobilized human telomere sequence probed by surface plasmon resonance using single-stranded DNA binding protein

Many sequences in genomic DNA are able to form unique tetraplex structures. Such structures are involved in a variety of important cellular processes and are emerging as a new class of therapeutic targets for cancers and other diseases. Screening for molecules targeting the tetraplex structure has been explored using such sequences immobilized on solid surfaces. Immobilized nucleic acids, in certain situations, may better resemble the molecules under in vivo conditions. In this report, we studied the formation of tetraplex structure of both the G-rich and C-rich strands of surface-immobilized human telomere sequence by surface plasmon resonance using the single-stranded DNA binding protein from Escherichia coli as probe. We demonstrate how the formation of G-quadruplex and i-motif could be probed under various conditions by this sequence-universal method. Our results also show that immobilization destabilized the tetraplex structure.     

Immobilization of single-stranded DNA by self-assembled polymer on gold substrate for a DNA chip

We developed a self-assembling polymer based on polyallylamine (PAH) for use in DNA chips. Thioctic acid (TA) was covalently attached to PAH in sidechains to immobilize the polymer on a gold surface by self-assembly. N-hydroxysuccinimide-ester terminated probe single-stranded (ss) DNA is easily covalently immobilized onto a TA-PAH-coated gold surface. Finally, the surface was covered with polyacrylic acid, which formed ion complexes with the TA-PAH, to reduce the cationic charge. This ssDNA on a polymer-coated surface recognized a fully matched DNA sequence and restrained nonspecific adsorption of target DNA. The selectivity and efficiency of hybridization was affected by adjusting the ionic strength of sodium chloride.     

Structural basis for promoter DNA recognition by the response regulator OmpR

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR–DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc–DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this “druggable” target.     

Structural basis for nick recognition by a minimal pluripotent DNA ligase

Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-A crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO4 nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a beta-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO4 termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.     

Structural basis for uracil recognition by archaeal family B DNA polymerases

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C-->A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.     

Structural basis for APPTPPPLPP peptide recognition by the FBP11WW1 domain

WW domains are small protein-protein interaction modules that recognize proline-rich stretches in proteins. The class II tandem WW domains of the formin binding protein 11 (FBP11) recognize specifically proteins containing PPLPp motifs as present in the formins that are involved in limb and kidney development, and in the methyl-CpG-binding protein 2 (MeCP2), associated with the Rett syndrome. The interaction involves the specific recognition of a leucine side-chain. Here, we report on the novel structure of the complex formed by the FPB11WW1 domain and the formin fragment APPTPPPLPP revealing the specificity determinants of class II WW domains.     

Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

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Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin

The crystal structure of a Man/Glc-specific lectin from the seeds of the bloodwood tree (Pterocarpus angolensis), a leguminous plant from central Africa, has been determined in complex with mannose and five manno-oligosaccharides. The lectin contains a classical mannose-specificity loop, but its metal-binding loop resembles that of lectins of unrelated specificity from Ulex europaeus and Maackia amurensis. As a consequence, the interactions with mannose in the primary binding site are conserved, but details of carbohydrate-binding outside the primary binding site differ from those seen in the equivalent carbohydrate complexes of concanavalin A. These observations explain the differences in their respective fine specificity profiles for oligomannoses. While Man(alpha1-3)Man and Man(alpha1-3)[Man(alpha1-6)]Man bind to PAL in low-energy conformations identical with that of ConA, Man(alpha1-6)Man is required to adopt a different conformation. Man(alpha1-2)Man can bind only in a single binding mode, in sharp contrast to ConA, which creates a higher affinity for this disaccharide by allowing two binding modes.     

Structural basis for the specific recognition of IL-18 by its alpha receptor

Interleukin 18 (IL-18), a member of the IL-1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand-binding primary receptor IL-18Rα and accessory receptor IL-18Rβ. Here we report the crystal structure of IL-18 with the ectodomain of IL-18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand-binding specificity of primary receptors in the IL-1 receptor family. We suggest that IL-18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand-receptor assembly and activation model for the IL-1 family.