Enzymatic Cleavage of 3'-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis.

The reversible dye-terminator (RDT)-based DNA sequencing-by-synthesis (SBS) chemistry has driven the advancement of the next-generation sequencing technologies for the past two decades. The RDT-based SBS chemistry relies on the DNA polymerase reaction to incorporate the RDT nucleotide (NT) for extracting DNA sequence information. The main drawback of this chemistry is the "DNA scar" issue since the removal of dye molecule from the RDT-NT after each sequencing reaction cycle leaves an extra chemical residue in the newly synthesized DNA. To circumvent this problem, we designed a novel class of reversible (2-aminoethoxy)-3-propionyl (Aep)-dNTPs by esterifying the 3'-hydroxyl group (3'-OH) of deoxyribonucleoside triphosphate (dNTP) and examined the NT-incorporation activities by A-family DNA polymerases. Using the large fragment of both Bacillus stearothermophilus (BF) and E. coli DNA polymerase I (KF) as model enzymes, we further showed that both proteins efficiently and faithfully incorporated the 3'-Aep-dNMP. Additionally, we analyzed the post-incorporation product of N + 1 primer and confirmed that the 3'-protecting group of 3'-Aep-dNMP was converted back to a normal 3'-OH after it was incorporated into the growing DNA chain by BF. By applying all four 3'-Aep-dNTPs and BF for an in vitro DNA synthesis reaction, we demonstrated that the enzyme-mediated deprotection of inserted 3'-Aep-dNMP permits a long, continuous, and scar-free DNA synthesis.

Thermococcus sp. 9°N DNA polymerase exhibits 3'-esterase activity that can be harnessed for DNA sequencing.

It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the Thermococcus sp. 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3'-editing based DNA sequencing method that could find practical applications after further optimization.

Structural Insights into Linear Tri-ubiquitin Recognition by A20-Binding Inhibitor of NF-κB, ABIN-2.

Recognition of linear polyubiquitin by specific ubiquitin-binding proteins plays an important role in mediating nuclear factor-κB (NF-κB) signaling. A20 binding proteins, ABINs, recognize linear polyubiquitin and A20 through UBAN and AHD1, respectively, for the inhibition of NF-κB activation. Here we report the crystal structure of the AHD1-UBAN fragment of ABIN2 in complex with linear tri-ubiquitin, which reveals a 2:1 stoichiometry of the complex. Structural analyses together with mutagenesis, pull-down, and isothermal titration calorimetry assays show that the hABIN2:tri-ubiquitin interaction is mainly through the primary ubiquitin-binding site, and also through the secondary ubiquitin-binding site under a high local protein concentration. Surprisingly, three ubiquitin units could form a right-handed helical trimer to bridge two ABIN2 dimers. The residues around the M1-linkage are crucial for ABIN2 to recognize tri-ubiquitin. The tri-ubiquitin bridging two ABIN2 dimers model suggests a possible higher-order signaling complex assembled between M1-linked polyubiquitinated proteins, ubiquitin-binding proteins, and effector signaling proteins in signal transduction.

Essential covalent linkage between the chymotrypsin-like domain and the extra domain of the SARS-CoV main protease.

The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.

Simultaneous production of trehalose, bioethanol, and high-protein product from rice by an enzymatic process.

Rice is a starch-rich raw material that can be used for trehalose production. It can be hydrolyzed with alpha-amylase, beta-amylase, and pullulanase to produce high-maltose content of rice saccharified solution for bioconversion of maltose into trehalose by trehalose synthase (TSase). For this purpose, an efficient enzymatic procedure has been successfully developed to simultaneously produce value-added trehalose, bioethanol, and high-protein product from rice as substrate. The highest maltose yield produced from the liquefied rice starch hydrolysate was 82.4 +/- 2.8% at 50 degrees C and pH 5.0 for 21-22 h. The trehalose conversion rate can reach at least 50% at 50 degrees C and pH 5.0 for 20-24 h by a novel thermostable recombinant Picrophilus torridus trehalose synthase (PTTS). All residual sugar, except trehalose, can be fully hydrolyzed by glucoamylase into glucose for further bioethanol production. The insoluble byproduct containing high yields of protein (75.99%) and dietary fiber (14.01%) can be processed as breakfast cereal product, health food, animal forage, etc. The conversion yield of bioethanol was about 98% after 64 h of fermentation time by Saccharomyces cerevisiae without any artificial culture solution addition. Ethanol can easily be separated from trehalose by distillation with a high recovery yield and purity of crystalline trehalose of 92.5 +/- 8.7% and 92.3%, respectively.