Euryarchaeal β-CASP Proteins with Homology to Bacterial RNase J Have 5′- to 3′-Exoribonuclease Activity

In the Archaea only a handful of ribonucleases involved in RNA processing and degradation have been characterized. One potential group of archaeal ribonucleases are homologues of the bacterial RNase J family, which have a β-CASP metallo-β-lactamase fold. Here we show that β-CASP proteins encoded in the genomes of the hyperthermophilic Euryarchaeota Pyrococcus abyssi and Thermococcus kodakaraensis are processive exoribonucleases with a 5′ end dependence and a 5′ to 3′ directionality. We named these enzymes Pab-RNase J and Tk-RNase J, respectively. RNAs with 5′-monophosphate or 5′-hydroxyl ends are preferred substrates of Pab-RNase J, whereas circularized RNA is resistant to Pab-RNase J activity. Degradation of a 3′ end-labeled synthetic RNA in which an internal nucleoside is substituted by three ethylene glycol units generates intermediates demonstrating 5′ to 3′ directionality. The substitution of conserved residues in Pab-RNase J predicted to be involved in the coordination of metal ions demonstrates their importance for ribonuclease activity, although the detailed geometry of the catalytic site is likely to differ from bacterial RNase J. This is the first identification of a 5′-exoribonuclease encoded in the genomes of the Archaea. Phylogenetic analysis shows that euryarchaeal RNase J has been inherited vertically, suggesting an ancient origin predating the separation of the Bacteria and the Archaea.     

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.     

Structural insights into catalysis and dimerization enhanced exonuclease activity of RNase?J

RNase J is a conserved ribonuclease that belongs to the β-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5′-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.     

Lack of sugar discrimination by human Pol mu requires a single glycine residue

DNA polymerase mu (Pol µ) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol µ is not able to discriminate against the 2′-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol µ is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol µ based on the available Pol β and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol µ was mutated to the consensus tyrosine present in Pol β, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol µ in DNA repair.     

Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-β-lactamase Superfamily

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-β-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and β-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-β-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.     

Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.     

Recognition of T-rich single-stranded DNA by the cold shock protein Bs-CspB in solution

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as ‘RNA chaperones’ and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT₇) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT₇ with only minor reorientations in loop β1–β2 and β3–β4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. ¹⁵N NMR relaxation and H/D exchange experiments revealed that binding of dT₇ increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop β3–β4.     

RNA aptamers selected against DNA polymerase beta inhibit the polymerase activities of DNA polymerases beta and kappa

DNA polymerase β (polβ), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polβ from a variable pool of 8 × 10¹² individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polβ activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polβ active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polβ also inhibited DNA polymerase κ, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polβ and κ, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase actvity in cells.     

Differential Effects of Tra2? Isoforms on HIV-1 RNA Processing and Expression

Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/ Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression.     

Low-fidelity DNA synthesis by human DNA polymerase theta

Human DNA polymerase theta (pol θ or POLQ) is a proofreading-deficient family A enzyme implicated in translesion synthesis (TLS) and perhaps in somatic hypermutation (SHM) of immunoglobulin genes. These proposed functions and kinetic studies imply that pol θ may synthesize DNA with low fidelity. Here, we show that when copying undamaged DNA, pol θ generates single base errors at rates 10- to more than 100-fold higher than for other family A members. Pol θ adds single nucleotides to homopolymeric runs at particularly high rates, exceeding 1% in certain sequence contexts, and generates single base substitutions at an average rate of 2.4 × 10⁻³, comparable to inaccurate family Y human pol κ (5.8 × 10⁻³) also implicated in TLS. Like pol κ, pol θ is processive, implying that it may be tightly regulated to avoid deleterious mutagenesis. Pol θ also generates certain base substitutions at high rates within sequence contexts similar to those inferred to be copied by pol θ during SHM of immunoglobulin genes in mice. Thus, pol θ is an exception among family A polymerases, and its low fidelity is consistent with its proposed roles in TLS and SHM.     

An Endoglucanase, EglA, from the Hyperthermophilic Archaeon Pyrococcus furiosus Hydrolyzes β-1,4 Bonds in Mixed-Linkage (1→3),(1→4)-β-d-Glucans and Cellulose

The eglA gene, encoding a thermostable endoglucanase from the hyperthermophilic archaeon Pyrococcus furiosus, was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 319-amino-acid protein with a calculated molecular mass of 35.9 kDa. The endoglucanase has a 19-amino-acid signal peptide but not cellulose-binding domain. The P. furiosus endoglucanase has significant amino acid sequence similarities, including the conserved catalytic nucleophile and proton donor, with endoglucanases from glucosyl hydrolase family 12. The purified recombinant enzyme hydrolyzed β-1,4 but not β-1,3 glucosidic linkages and had the highest specific activity on cellopentaose (degree of polymerization [DP] = 5) and cellohexaose (DP = 6) oligosaccharides. To a lesser extent, EglA also hydrolyzed shorter cellodextrins (DP < 5) as well as the amorphous portions of polysaccharides which contain only β-1,4 bonds such as carboxymethyl cellulose, microcrystalline cellulose, Whatman paper, and cotton linter. The highest specific activity toward polysaccharides occurred with mixed-linkage β-glucans such as barley β-glucan and lichenan. Kinetics studies with cellooliogsaccharides and p-nitrophenyl-cellooligosaccharides indicated that the enzyme had three glucose binding subsites (−I, −II, and −III) for the nonreducing end and two glucose binding subsites (+I and +II) for the reducing end from the scissile glycosidic linkage. The enzyme had temperature and pH optima of 100°C and 6.0, respectively; a half-life of 40 h at 95°C; and a denaturing temperature of 112°C as determined by differential scanning calorimetry. The discovery of a thermostable enzyme with this substrate specificity has implications for both the evolution of enzymes involved in polysaccharide hydrolysis and the occurrence of growth substrates in hydrothermal vent environments.     

The Atomic Resolution Structure of Human AlkB Homolog 7 (ALKBH7), a Key Protein for Programmed Necrosis and Fat Metabolism

ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … X_n … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the “nucleotide recognition lid” which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.     

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]₂, in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]₂ as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]₂. Long-lived water molecules with correlation time τ_c longer than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA–DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA–DNA junction adopts an O4′-endo sugar conformation, while the other DNA residues including 3C in the DNA–RNA junction, adopt C1′-exo or C2′-endo conformations. The exchange rates for RNA C2′-OH were found to be ~5–20 s^–1. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂ is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.     

Sequence and structural analysis of 4SNc-Tudor domain protein from Takifugu Rubripes

The fugu SN4TDR protein belongs to an evolutionarily conserved family, consisting of four repeat staphylococcal nuclease-like domains (SN1-SN4) at the N-terminus followed by Tudor and SN-like domains (TSN). Sequence analysis showed that the C-terminal TSN domain is composed of a complete SN-like domain interdigitated with a Tudor domain. In despite of low level of sequence identities, five SN-like domains have a few conserved amino acids that may play essential roles in the function of the protein. Computer modeling and secondary structural prediction of the SN-like domains revealed the presence of similar structural features of β1-β2-β3-α1-β4-β5-α2-α3, which provides a structural basis for oligonucleotides binding. The loop region L_3α for binding sites between β3 and α1 of SN-like domains are different from human p100, implying the divergence in the structures of binding sites. These results indicate that fugu SN4TDR may bind methylated ligands and/or oligonucleotides through its distant domains.