Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants.

Allele-specific PCR (AS-PCR) has been used as a simple, cost-effective method for genotyping and gene mapping in research and clinical settings. AS-PCR permits the detection of single nucleotide variants and insertion or deletion variants owing to the selective extension of a perfectly matched primer (to the template DNA) over a mismatched primer. Thus, the mismatch discrimination power of the DNA polymerase is critical. Unfortunately, currently available polymerases often amplify some mismatched primer-template complexes as well as matched ones, obscuring AS detection. To increase mismatch discrimination, mutations were generated in the Thermus aquaticus (Taq) DNA polymerase, the most efficient variant was selected, and its performance evaluated in single nucleotide polymorphism and cancer mutation genotyping. In addition, the primer design and reaction buffer conditions were optimized for AS amplification. Our highly selective AS-PCR, which is based on an allele-discriminating priming system that leverages a Taq DNA polymerase variant with optimized primers and reaction buffer, can detect mutations with a mutant allele frequency as low as 0.01% in genomic DNA and 0.0001% in plasmid DNA. This method serves as a simple, fast, cost-effective, and ultra-sensitive way to detect single nucleotide variants and insertion or deletion mutations with low abundance.

Synergistic Effect of Detection and Separation for Pathogen Using Magnetic Clusters.

Early diagnosis of infectious diseases is important for treatment; therefore, selective and rapid detection of pathogenic bacteria is essential for human health. We report a strategy for highly selective detection and rapid separation of pathogenic microorganisms using magnetic nanoparticle clusters. Our approach to develop probes for pathogenic bacteria, including Salmonella, is based on a theoretically optimized model for the size of clustered magnetic nanoparticles. The clusters were modified to provide enhanced aqueous solubility and versatile conjugation sites for antibody immobilization. The clusters with the desired magnetic property were then prepared at critical micelle concentration (CMC) by evaporation-induced self-assembly (EISA). Two different types of target-specific antibodies for H- and O-antigens were incorporated on the cluster surface for selective binding to biological compartments of the flagella and cell body, respectively. For the two different specific binding properties, Salmonella were effectively captured with the O-antibody-coated polysorbate 80-coated magnetic nanoclusters (PCMNCs). The synergistic effect of combining selective targeting and the clustered magnetic probe leads to both selective and rapid detection of infectious pathogens.

Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates.

The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a "5' end binding pocket" that hosts the 5' nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3' flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5' flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3' flap DNA substrates with 5' nicked ends.

Apo and InsP3-bound crystal structures of the ligand-binding domain of an InsP3 receptor.

We report the crystal structures of the ligand-binding domain (LBD) of a rat inositol 1,4,5-trisphosphate receptor (InsP(3)R) in its apo and InsP(3)-bound conformations. Comparison of these two conformations reveals that LBD's first ß-trefoil fold (ß-TF1) and armadillo repeat fold (ARF) move together as a unit relative to its second ß-trefoil fold (ß-TF2). Whereas apo LBD may spontaneously transition between gating conformations, InsP(3) binding shifts this equilibrium toward the active state.

Structure-function study of the N-terminal domain of exocyst subunit Sec3.

The exocyst is an evolutionarily conserved octameric complex involved in polarized exocytosis from yeast to humans. The Sec3 subunit of the exocyst acts as a spatial landmark for exocytosis through its ability to bind phospholipids and small GTPases. The structure of the N-terminal domain of Sec3 (Sec3N) was determined ab initio and defines a new subclass of pleckstrin homology (PH) domains along with a new family of proteins carrying this domain. Respectively, N- and C-terminal to the PH domain Sec3N presents an additional alpha-helix and two beta-strands that mediate dimerization through domain swapping. The structure identifies residues responsible for phospholipid binding, which when mutated in cells impair the localization of exocyst components at the plasma membrane and lead to defects in exocytosis. Through its ability to bind the small GTPase Cdc42 and phospholipids, the PH domain of Sec3 functions as a coincidence detector at the plasma membrane.

Large nucleotide-dependent conformational change in Rab28.

Rab GTPases are essential regulators of membrane trafficking. We report crystal structures of Rab28 in the active (GppNHp-bound) and inactive (GDP-3'P-bound) forms at 1.5 and 1.1A resolution. Rab28 is a distant member of the Rab family. While the overall fold of Rab28 resembles that of other Rab GTPases, it undergoes a larger nucleotide-dependent conformational change than other members of this family. Added flexibility resulting from a double-glycine motif at the beginning of switch 2 might partially account for this observation. The double-glycine motif, which is conserved in the Arf family, only occurs in Rab28 and Rab7B of the Rab family, and may have a profound effect on their catalytic activities.

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding.

On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-beta-actin and profilin-alpha-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7 degrees rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.

Crystal structure of human cyclin K, a positive regulator of cyclin-dependent kinase 9.

Cyclin K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 A resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27(Kip1). Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes.