Mechanisms of Mutagenesis by a Bulky DNA Lesion at the Guanine N7 Position

The RpII215 locus encodes the large subunit of RNA polymerase II (polII). Three of 22 RpII215 alleles cause a synergistic enhancement of the mutant phenotype elicited by mutations in the Ultrabithorax (Ubx) locus. We have recovered and analyzed three new mutations that suppress this enhancement. All three mutations map to the RpII215 locus. In addition to suppressing the Ubx enhancement of other RpII215 alleles, two of the new mutations, JH1 and WJK2, themselves enhance Ubx. RpII215 alleles can be placed into three classes based on their ability to enhance Ubx. Class I alleles, including Ubl, C4, C11, JH1, and WJK2, enhance Ubx when heterozygous with class II alleles, which include wild-type RpII215. Class III alleles, which include amorphic alleles, do not enhance Ubx. The third new mutation, WJK1, is a conditional amorphic allele, which behaves like a class III allele at 29 degrees but like a class II allele at 19 degrees. Another mutant phenotype is caused by certain RpII215 alleles, including all class I alleles. This phenotype is a synergistic enhancement of a mutant phenotype elicited by mutations at the Delta (Dl) locus. Unlike the enhancement of Ubx, the enhancement of Dl is not dependent upon antagonistic interactions between different classes of RpII215 alleles.     

Guanine and 7-methylguanine amino proton exchange rates as a function of buffer pK: implications for the exchange mechanism.

Using the stopped-flow kinetic method we have measured the deuteration rate of the amino protons in 2'deoxyguanosine 5'monophosphate and 7-methylguanosine 5'monophosphate. For both compounds the exchange rates are accelerated with increasing concentration of a large number of buffers with widely differing pKs. The results obtained, in conjunction with a theoretical model study, give rise to serious doubts concerning the normally accepted mechanism of amino proton exchange involving a pre-protonation at N7.     

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Exploring purine N7 interactions via atomic mutagenesis: the group I ribozyme as a case study

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Evolution of the Hemagglutinin Protein of the New Pandemic H1N1 Influenza Virus: Maintaining Optimal Receptor Binding by Compensatory Substitutions

Pandemic influenza A H1N1 (pH1N1) virus emerged in 2009. In the subsequent 4 years, it acquired several genetic changes in its hemagglutinin (HA). Mutations may be expected while virus is adapting to the human host or upon evasion from adaptive immune responses. However, pH1N1 has not displayed any major antigenic changes so far. We examined the effect of the amino acid substitutions found to be most frequently occurring in the pH1N1 HA protein before 1 April 2012 on the receptor-binding properties of the virus by using recombinant soluble HA trimers. Two changes (S186P and S188T) were shown to increase the receptor-binding avidity of HA, whereas two others (A137T and A200T) decreased binding avidity. Construction of an HA protein tree revealed the worldwide emergence of several HA variants during the past few influenza seasons. Strikingly, two major variants harbor combinations of substitutions (S186P/A137T and S188T/A200T, respectively) with opposite individual effects on binding. Stepwise reconstruction of the HA proteins of these variants demonstrated that the mutations that increase receptor-binding avidity are compensated for by the acquisition of subsequent mutations. The combination of these substitutions restored the receptor-binding properties (avidity and specificity) of these HA variants to those of the parental virus. The results strongly suggest that the HA of pH1N1 was already optimally adapted to the human host upon its emergence in April 2009. Moreover, these results are in agreement with a recent model for antigenic drift, in which influenza A virus mutants with high and low receptor-binding avidity alternate.     

Specificity of Lysine:N-Hydroxylase: A Hypothesis for a Reactive Substrate Intermediate in the Catalytic Mechanism

The recombinant cytoplasmic preparation of lysine:N⁶-hydroxlase catalyzes the conversion ofL-lysine to itsN⁶-hydroxy derivative when supplemented with the cofactors NADPH and FAD. A number of lysine analogs reflecting minor alterations in the inherent structural features of the amino acid as well compounds with relatively high affinity for lysine binding domains in other proteins were examined for their ability to serve as substrates of lysine:N⁶-hydroxylase. These studies have revealed that the enzyme does not tolerate any change in the structural features ofL-lysine, its preferred substrate, with the exception of the replacement of the C_γH₂-methylene group by sulfur, as in (S)-2-aminoethyl-L-cysteine.L-Norleucine is a potent inhibitor of the enzyme whileL-norvaline andL-α-aminobutyric acid do not exhibit such effect, indicating the importance of a C₄hydrophobic side chain for effective interaction with the enzyme. Among theN-alkyl amides of hydrophobic amino acids, onlyL-norleucine methylamide andL-α-aminobutyric acid ethylamide serve as moderate inhibitors of lysine:N⁶-hydroxylase. Based on the enzyme's stringent substrate specificity, a mechanism involving the conversion ofL-lysine to 2-aminocaprolactam prior to its oxygenation by the 4a-peroxyflavin intermediate in the catalytic cycle is proposed.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.     

靶向VEGF小干扰RNA协同5-FU诱导人乳腺癌细胞MCF-7凋亡机制的研究

探讨慢病毒介导的靶向VEGF小干扰RNA联合应用化疗药物5 FU诱导人乳腺癌细胞MCF-7凋亡的机制。以携带VEGF siRNA的慢病毒载体感染MCF-7细胞,应用RT-PCR和Western blot分别检测各组VEGF mRNA、VEGF蛋白及凋亡相关蛋白的表达,流式细胞术检测细胞凋亡。结果表明,慢病毒VEGF siRNA干扰组细胞VEGF mRNA和蛋白表达水平明显低于对照组,凋亡相关蛋白P53及P21表达上调,而SIRT1、Bcl-2及Survivin表达下调。流式细胞术检测显示慢病毒干扰组及5-FU组细胞凋亡率显著升高,联合治疗组的协同作用更为明显。上述结果表明：慢病毒介导的RNA干扰能明显抑制MCF-7细胞VEGF的表达,通过下调SIRTI蛋白的表达,导致P53蛋白表达上调,并调控其下游P21、bcl-2和Survivin的表达,从而诱导MCF-7细胞的凋亡,并且提高了MCF-7对5-FU的敏感性。     

Formation of a phosphoramide mustard-nucleotide adduct that is not by alkylation at the N7 position of guanine

The reaction of 2'-deoxyguanosine 3'-monophosphate with phosphoramide mustard resulted in the formation of several adducts. One of these adducts was formed by linking phosphoramide mustard to the phosphate group of 2'-deoxyguanosine 3'-monophosphate rather than by the generally accepted mechanism involving alkylation at the N7 position of guanine. This adduct served as an acceptor for the transfer of 32p from [gamma 32P]ATP by polynucleotide kinase and thus could be detected by the sensitive 32p-postlabeling assay.     

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.     

Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and in virus biology

We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.     

Alginate Lyase Aly36B is a New Bacterial Member of the Polysaccharide Lyase Family 36 and Catalyzes by a Novel Mechanism With Lysine as Both the Catalytic Base and Catalytic Acid

Alginate lyases, which are important in both basic and applied sciences, fall into ten polysaccharide lyase (PL) families. PL36 is a newly established family that includes 39 bacterial sequences and one eukaryotic sequence. Till now, the structures or catalytic mechanisms of PL36 alginate lyases have yet to be revealed. Here, we characterized a novel PL36 alginate lyase, Aly36B, from Chitinophaga sp. MD30. Aly36B is a polymannuronate specific endolytic alginate lyase. To probe the catalytic mechanism of Aly36B, the structures of wild-type Aly36B and its mutants (K143A/Y185A in complex with alginate tetrasaccharide and K143A/M171A with trisaccharide) were solved. The overall structure of Aly36B belongs to the β-jelly roll scaffold, adopting a typical β-sandwich fold. Aly36B contains a Ca²⁺, which is far away from the active center and plays an important role in stabilizing the structure of Aly36B. Based on structural and mutational analyses, the catalytic mechanism of Aly36B for alginate degradation was explained. During catalysis, Arg¹⁶⁹, Tyr¹⁸⁵, and Tyr¹⁸⁷ are responsible for neutralizing the negative charge of the substrate, and Lys¹⁴³ acts as both the catalytic base and the catalytic acid, which represents a new kind of catalytic mechanism of alginate lyases. Sequence alignment shows that these four residues involved in catalysis are highly conserved in all PL36 sequences, suggesting that PL36 alginate lyases may adopt a similar catalytic mechanism. Taken together, this study reveals the molecular structure and catalytic mechanism of a PL36 alginate lyase, broadening our knowledge on alginate lyases and facilitating future biotechnological applications of PL36 alginate lyases.     

From Induced Fit to Conformational Selection: A Continuum of Binding Mechanism Controlled by the Timescale of Conformational Transitions

In receptor-ligand binding, a question that generated considerable interest is whether the mechanism is induced fit or conformational selection. This question is addressed here by a solvable model, in which a receptor undergoes transitions between active and inactive forms. The inactive form is favored while unbound but the active form is favored while a ligand is loosely bound. As the active-inactive transition rates increase, the binding mechanism gradually shifts from conformational selection to induced fit. The timescale of conformational transitions thus plays a crucial role in controlling binding mechanisms.