A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Characterization of the direct physical interaction of nc886, a cellular non-coding RNA, and PKR

We have recently shown that nc886 (pre-miR-886 or vtRNA2-1) is not a genuine microRNA precursor nor a vault RNA, but a novel type of non-coding RNA that represses PKR, a double-stranded RNA (dsRNA) dependent kinase. Here we have characterized their direct physical association. PKR’s two RNA binding domains form a specific and stable complex with nc886’s central portion, without any preference to its 5′-end structure. By binding to PKR with a comparable affinity, nc886 competes with dsRNA and attenuates PKR activation by dsRNA. Our data suggest that nc886 sets a threshold for PKR activation so that it occurs only during genuine viral infection but not by a minute level of fortuitous cellular dsRNA.     

Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway.One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H₂O₂ and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N⁶-ethenoadenine (ɛA) and 3,N⁴-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H₂O₂ or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway.     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

MvnI: A restriction enzyme in the archaebacterium Methanococcus vannielii

Abstract The methanogenic archaebacerium Methanococcus vannielii contains a type II restriction endonuclease. The enzyme was purified by a simple three-step procedure resulting in enzyme preparations free of contaminating unspecific nucleases. The restriction enzyme recognizes and cleaves the sequence 5'-CG ↓ CG-3' ( Fnu DII and Tha I isoschizomer) and generates DNA fragments with blunt ends. Due to its purity and activity at moderate temperatures, Mvn I might be a useful alternative to Fnu DII and Tha I active at 60°C.     

Multi-domain Packing in the Aminoacylatable 3′ End of a Plant Viral RNA

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure (TLS) in its 3′ untranslated region (3′ UTR).  This highly structured element induces valylation of the viral RNA by host cell enzymes and is important for virus proliferation. Directly upstream of the TYMV TLS is an upstream pseudoknot domain (UPD) that has been considered to be structurally distinct from the TLS.  However, using a combination of functional, biochemical, and biophysical assays, we show that the entire 3′ UTR of the viral genome is a single structured element in the absence of cellular protein.  This packing architecture stabilizes the RNA structure and creates a better substrate for aminoacylation, and thus the UPD and TLS are functionally and structurally coupled.  It has been proposed that the TYMV TLS acts as a molecular switch between translation and replication. Our results suggest that this putative switch could be based on structural changes within the global architecture of the UTR induced by interactions with the ribosome. The TYMV TLS·UPD might demonstrate how RNA structural plasticity can play a role in regulation of biological processes.     

Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils

We present site-directed chemical modification (SDCM), a tool for engineering U-resistant archaeal DNA polymerases of family B. The Thermococcus litoralis Sh1B DNA polymerase (GenBank: GQ891548) was chosen as the object of the study. Similar to D.Tok, Kod1, Pfu, Tgo and other archaeal members of this family, the T. litoralis Sh1B DNA polymerase is a domain structured, proofreading-proficient enzyme that has the polymerization and 3′→5′ DNA exonucleolytic activities and contains N-terminally located highly conserved template-strand U-binding pocket. The tight binding of template uracil in the enzyme pocket during polymerization blocks the replication of DNA containing uracils. This effect can be alleviated by mutations in key amino acids of the U-binding pocket. We altered T. litoralis Sh1B DNA polymerase's ability to read through the template-strand uracils by applying SDCM. Specific modification of individual cysteine residues in U-binding pocket — targets introduced into certain positions by site-directed mutagenesis — enables the enzyme to effectively replicate DNA containing uracils. We demonstrate that the acquired resistance of chemically modified T. litoralis Sh1B DNA polymerase to DNA uracil correlates with its decreased affinity for template-strand uracil.     

Functional characterization of the Saccharomyces cerevisiae ABC-transporter Yor1p overexpressed in plasma membranes

Yor1p, a Saccharomyces cerevisiae plasma membrane ABC-transporter, is associated to oligomycin resistance and to rhodamine B transport. Here, by using the overexpressing strain Superyor [A. Decottignies, A.M. Grant, J.W. Nichols, H. de Wet, D.B. McIntosh, A. Goffeau, ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p, J. Biol. Chem. 273 (1998) 12612-12622], we show that Yor1p also confers resistance to rhodamine 6G and to doxorubicin. In addition, Yor1p protects cells, although weakly, against tetracycline, verapamil, eosin Y and ethidium bromide. The basal ATPase activity of the overexpressed form of Yor1p was studied in membrane preparations. This activity is quenched upon addition of micromolar amounts of vanadate. V_max and K_m values of ∼ 0.8 s^− 1 and 50 ± 8 μM are measured. Mutations of essential residues in the nucleotide binding domain 2 reduces the activity to that measured with a Δyor1 strain. ATP hydrolysis is strongly inhibited by the addition of potential substrates of the transporter. Covalent reaction of 8-azido-[α-³²P]ATP with Yor1p is not sensitive to the presence of excess oligomycin. Thus, competition of the drug with ATP binding is unlikely. Finally, we inspect possible hypotheses accounting for substrate inhibition, rather than stimulation, of ATP hydrolysis by the membrane preparation.     

Crystallographic Analysis of the Reaction Cycle of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase,a Unique Member of the 2H Phosphoesterase Family

2H phosphoesterases catalyze reactions on nucleotide substrates and contain two conserved histidine residues in the active site. Very limited information is currently available on the details of the active site and substrate/product binding during the catalytic cycle of these enzymes. We performed a comprehensive X-ray crystallographic study of mouse 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a membrane-associated enzyme present at high levels in the tetrapod myelin sheath. We determined crystal structures of the CNPase phosphodiesterase domain complexed with substrate, product, and phosphorothioate analogues. The data provide detailed information on the CNPase reaction mechanism, including substrate binding mode and coordination of the nucleophilic water molecule. Linked to the reaction, an open/close motion of the β5–α7 loop is observed. The role of the N terminus of helix α7—unique for CNPase in the 2H family—during the reaction indicates that 2H phosphoesterases differ in their respective reaction mechanisms despite the conserved catalytic residues. Furthermore, based on small-angle X-ray scattering, we present a model for the full-length enzyme, indicating that the two domains of CNPase form an elongated molecule. Finally, based on our structural data and a comprehensive bioinformatics study, we discuss the conservation of CNPase in various organisms.