R2D ligase: Unveiling a novel DNA ligase with surprising DNA-to-RNA ligation activity.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5' phosphates and 3' hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double-stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acid substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Cronobacter phage CR 9 (R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5' and 3' ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA-to-DNA or DNA-to-RNA. The ligation of a DNA strand to the 5'-PO4 2- end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and the preparation of chimeric guide DNA-RNA for gene editing applications.

Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases.

BACKGROUND: The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called 'Point-of-Care tests'. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited. RESULTS: We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8-9 and 25-110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25-37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes. CONCLUSIONS: Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity.

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity. The enzyme is more heat-labile than its counterpart from E. coli and exists as a homodimer in solution. The crystal structure of the enzyme has been determined to a resolution of 3.15 Å, indicating an important role of a disulfide bridge for the homodimer formation and as such for the function of MG Orn. Substitution of the Cys110 residue with either Gly or Ala hampered the dimer formation and severely affected the enzyme's ability to act on RNA. A conserved loop containing His128-Tyr129-Arg130 in the neighboring monomer is probably involved in efficient binding and processing of longer RNA substrates than diribonucleotides.

Structure of the X (ADRP) domain of nsp3 from feline coronavirus.

The structure of the X (or ADRP) domain of a pathogenic variant of feline coronavirus (FCoV) has been determined in tetragonal and cubic crystal forms to 3.1 and 2.2 A resolution, respectively. In the tetragonal crystal form, glycerol-3-phosphate was observed in the ADP-ribose-binding site. Both crystal forms contained large solvent channels and had a solvent content of higher than 70%. Only very weak binding of this domain to ADP-ribose was detected in vitro. However, the structure with ADP-ribose bound was determined in the cubic crystal form at 3.9 A resolution. The structure of the FCoV X domain had the expected macro-domain fold and is the first structure of this domain from a coronavirus belonging to subgroup 1a.

Crystal structures of the X-domains of a Group-1 and a Group-3 coronavirus reveal that ADP-ribose-binding may not be a conserved property.

The polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (Nsps). Consisting of five domains, Nsp3 is the largest of these (180-210 kDa). Among these domains, the so-called X-domain is believed to act as ADP-ribose-1''-phosphate phosphatase or to bind poly(ADP-ribose). However, here we show that the X-domain of Infectious Bronchitis Virus (strain Beaudette), a Group-3 coronavirus, fails to bind ADP-ribose. This is explained on the basis of the crystal structure of the protein, determined at two different pH values. For comparison, we also describe the crystal structure of the homologous X-domain from Human Coronavirus 229E, a Group-1 coronavirus, which does bind ADP-ribose.

Production of coronavirus nonstructural proteins in soluble form for crystallization.

For biophysical investigations on viral proteins, in particular for structure determination by X-ray crystallography, relatively large quantities of purified protein are necessary. However, expression of cDNAs coding for viral proteins in prokaryotic or eukaryotic systems is often not straightforward, and frequently the amount and/or the solubility of the protein obtained are not sufficient. Here, we describe a number of protocols for production of nonstructural proteins of coronaviruses that have proven to be efficient in increasing expression yields or solubilities.