Free Uracil Research Articles

Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3 (BE3) and targeted activation-induced cytidine deaminase (CDA) (target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase (UDG) inhibitor (UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a high-fidelity and high-efficiency solution for rice genomic base editing.

Nucleosides 10: Synthesis of New Derivatives of Pyrimidine and Fused Pyrimidine Nucleosides of Expected Biological Activity

Pyrimidines, such as 6-amino-2-thio and 2-methylthiouracils and fused pyrimidines, such as thienopyrimidines reacted with 1-O-acetyl-2,3,5-tri-O- benzoyl-β-D-ribofuranose to get new derivatives of the corresponding nucleosides. The obtained protected nucleosides were deprotected by methanolic sodium methoxide to get the corresponding free uracil and thienopyrimidine nucleosides. The new nucleosides formed were tested for biological activity against some of microorganism (some fungi and bacteria species). Some of the tested products showed moderate activity and the results were reported.

Product release mechanism and the complete enzyme catalysis cycle in yeast cytosine deaminase (yCD): A computational study

Yeast cytosine deaminase (yCD) is critical in gene-directed enzyme prodrug therapy as it catalyzes the hydrolytic deamination of cytosine. The product (uracil) release process is considered as rate-limiting in the whole enzymatic catalysis and includes the cleavage of the uracil-metal bond and the delivery of free uracil out of the reactive site. Herein extensive combined random acceleration molecular dynamics (RAMD) and molecular dynamics (MD) simulations coupled with the umbrella sampling technique have been performed to study the product transport mechanism. Five channels have been identified, and the thermodynamic and dynamic characterizations for the two most favorable channels have been determined and analyzed. The free energy barrier for the most beneficial pathway is about 13kcal/mol and mainly results from the cleavage of hydrogen bonds between the ligand uracil and surrounding residues Asn51, Glu64, and Asp155. The conjugated rings of Phe114 and Trp152 play gating and guiding roles in the product delivery via π⋯π van der Waals interactions with the product. Finally, the full cycle of the enzymatic catalysis has been determined, making the whole process computationally more precise.

Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

The Escherichia coli very short patch (VSP) repair pathway corrects thymidine-guanine mismatches that result from spontaneous hydrolytic deamination damage of 5-methyl cytosine. The VSP repair pathway requires the Vsr endonuclease, DNA polymerase I, a DNA ligase, MutS, and MutL to function at peak efficiency. The biochemical roles of most of these proteins in the VSP repair pathway have been studied extensively. However, these proteins have not been studied together in the context of VSP repair in an in vitro system. Using purified components of the VSP repair system in a reconstitution reaction, we have begun to develop an understanding of the role played by each of these proteins in the VSP repair pathway and have gained insights into their interactions. In this report we demonstrate an in vitro reconstitution of the VSP repair pathway using a plasmid DNA substrate. Surprisingly, the repair track length can be modulated by the concentration of DNA ligase. We propose roles for MutL and MutS in coordination of this repair pathway.

Electron Affinities of Uracil: Microsolvation Effects and Polarizable Continuum Model

We present adiabatic electron affinities (AEAs) and the vertical detachment energies (VDEs) of the uracil molecule interacting with one to five water molecules. Credibility of MP2 and DFT/B3LYP calculations is supported by comparison with available benchmark CCSD(T) data. AEAs and VDEs obtained by MP2 and DFT/B3LYP methods copy trends of benchmark CCSD(T) results for the free uracil and uracil-water complexes in the gas phase being by 0.20 - 0.28 eV higher than CCSD(T) values depending on the particular structure of the complex. AEAs and VDEs from MP2 are underestimated by 0.09-0.15 eV. For the free uracil and uracil-(H(2)O)(n) (n = 1,2,3,5) complexes, we also consider the polarizable continuum model (PCM) and discuss the importance of the microsolvation when combined with PCM. AEAs and VDEs of uracil and uracil-water complexes enhance rapidly with increasing relative dielectric constant (ε) of the solvent. Highest AEAs and VDEs of the U(H(2)O)(5) complexes from B3LYP with ε = 78.4 are 2.03 and 2.81 eV, respectively, utilizing the correction from CCSD(T). Specific structural features of the microsolvated uracil-(H(2)O)(n) complexes and their anions are preserved also upon considering PCM in calculations of AEAs and VDEs.

Excited electronic states and photophysics of uracil–water complexes

The electronically excited singlet states of complexes of uracil with one water molecule have been studied theoretically using ab initio multireference configuration interaction methods. In agreement with previous theoretical and experimental results, four cyclic isomers of uracil forming hydrogen bonds with the water molecule have been located with energies within 0.2 eV from the lowest energy isomer. Focus has been given on the mechanism for radiationless decay to the ground state after initial UV absorption and on the effect of complexation with water on previously reported radiationless decay pathways. Features on the excited state potential energy surfaces, such as minima, transition states and conical intersections, have been located for all isomers and compared with those of free uracil. The hydrogen-bonded water molecule changes the relative energies of these features and may lead to different excited state dynamics and lifetimes, in agreement with experimental observations.

Cloning, expression, and characterization of uracil-DNA glycosylase of Chlamydia pneumoniae in Escherichia coli

A uracil-DNA glycosylase gene was cloned from Chlamydia pneumoniae AR39 and expressed in E. coli strains BL21 (DE3) and BL21 (DE3) pLysS. After purification by Ni–NTA His · Bind Resin and DEAE Sepharose Fast Flow column chromatography, recombinant CpUDG with a specific activity of 1,000,000 U/mg was obtained. The enzymatic activity of the purified CpUDG protein was further characterized using oligodeoxyribonucleotides carrying uracil bases as substrates. The base opposite to uracil in double strand DNAs affected uracil removal efficiencies in the order: U/– > U/T > U/C > U/G > U/A. Free uracil and abasic sites (AP site) could inhibit the reaction. The optimal temperature and pH for uracil removal by CpUDG were 37 °C and pH 8.0, respectively. Site-directed mutagenesis studies indicated that amino acids D77, H200, and A205 were important for the catalytic activity of CpUDG. Together, these data suggest that CpUDG is a member of the UDG family-I protein. This is the first report on cloning, expression, and characterization of Chlamydia uracil-DNA glycosylase.

Theoretical study of hydrogenation of thiouracils and their base pairs with adenine

AbstractThe purpose of the present work is to employ theoretical chemical tools to explore the subtle features of the hydrogenation of uracil and its related thioderivatives. The reactions of hydrogenation are followed for the free uracil, 2‐thiouracil, 4‐thiouracil, and 2,4‐dithiouracil as well as for adenine–thiouracil base pairs. It is shown for the first time that, in contrast to the hydrogenation pathways to the carbon atoms C5 and C6 of the studied molecules, characterized by the transition states, the hydrogenations at the sulphur atoms for thiouracils are barrierless and thus the latter are the kinetically most favorable pathways for hydrogenation. As also shown, these features are retained under the hydrogenation of the adenine–thiouracil base pairs. The present study clearly demonstrates that the base pairing destruction is the key radiation‐induced lesion in the adenine–4‐thiouracil and adenine–2,4‐dithiouracil base pairs when the hydrogen radical H·, as one of the primary radiation products, resides at the sulphur atom S4. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2004

The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features.

Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9 A resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63 A. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG.

Raman spectroscopy of uracil DNA glycosylase-DNA complexes: insights into DNA damage recognition and catalysis.

Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG). The binding of undamaged DNA to UDG results in decreased intensity of the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2'-beta-fluorodeoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-)(1) downshifts in the carbonyl normal modes. Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouridine analogue such that a change in hybridization at C1 occurs [Parikh, S. S., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueous solution, contributing significantly to catalysis. These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.

Crystal structure ofEscherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 A), its complex with uracil (1.50 A), and a second active-site complex with glycerol (1.43 A). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of k(cat) for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results.

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single-stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high-resolution crystal structures of free UDG from Escherichia coli strain B (1.60 Å), its complex with uracil (1.50 Å), and a second active-site complex with glycerol (1.43 Å). These represent the first high-resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side-chain positions of the putative general-acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active-site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate-induced conformational changes to bring active-site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of kcat for wild-type UDG and the D64N and H187Q mutant enzymes is consistent with general-base catalysis by Asp64, but provides no evidence for a general-acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results. Proteins 1999;35:13–24. © 1999 Wiley-Liss, Inc.

Hydration structure of uracil as studied by 1D and 2D heteronuclear Overhauser spectroscopy: evidence for the formation of a trihydrate in the first solvation shell

The intermolecular heteronuclear Overhauser effect (HOE) between water and uracil has been measured. Steady-state 13C, 1H nuclear Overhauser effect (NOE) results show that the first solvation shell includes three water molecules. Transient 13C, 1H and 15N, 1H 2D HOESY experiments as well as 1H NOESY experiments give further evidence for the formation of a cyclic trihydrate as obtained by three bridge hydrogen-bonding between the three water molecules and the two amide groups of the uracil. The trihydrate has only a very short life-time as compared to the rotational correlation time of the free uracil molecule which remains the tumbling species in the water–DMSO solution.

A Sequence in the N-terminal Region of Human Uracil-DNA Glycosylase with Homology to XPA Interacts with the C-terminal Part of the 34-kDa Subunit of Replication Protein A

Uracil-DNA glycosylase releases free uracil from DNA and initiates base excision repair for removal of this potentially mutagenic DNA lesion. Using the yeast two-hybrid system, human uracil-DNA glycosylase encoded by the UNG gene (UNG) was found to interact with the C-terminal part of the 34-kDa subunit of replication protein A (RPA2). No interaction with RPA4 (a homolog of RPA2), RPA1, or RPA3 was observed. A sandwich enzyme-linked immunosorbent assay with trimeric RPA and the two-hybrid system both demonstrated that the interaction depends on a region in UNG localized between amino acids 28 and 79 in the open reading frame. In this part of UNG a 23-amino acid sequence has a significant homology to the RPA2-binding region of XPA, a protein involved in damage recognition in nucleotide excision repair. Trimeric RPA did not enhance the activity of UNG in vitro on single- or double-stranded DNA. A part of the N-terminal region of UNG corresponding in size to the complete presequence was efficiently removed by proteinase K, leaving the proteinase K-resistant compact catalytic domain intact and fully active. These results indicate that the N-terminal part constitutes a separate structural domain required for RPA binding and suggest a possible function for RPA in base excision repair.

A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA.

Any uracil bases in DNA, a result of either misincorporation or deamination of cytosine, are removed by uracil-DNA glycosylase (UDG), one of the most efficient and specific of the base-excision DNA-repair enzymes. Crystal structures of human and viral UDGs complexed with free uracil have indicated that the enzyme binds an extrahelical uracil. Such binding of undamaged extrahelical bases has been seen in the structures of two bacterial methyltransferases and bacteriophage T4 endonuclease V. Here we characterize the DNA binding and kinetics of several engineered human UDG mutants and present the crystal structure of one of these, which to our knowledge represents the first structure of any eukaryotic DNA repair enzyme in complex with its damaged, target DNA. Electrostatic orientation along the UDG active site, insertion of an amino acid (residue 272) into the DNA through the minor groove, and compression of the DNA backbone flanking the uracil all result in the flipping-out of the damaged base from the DNA major groove, allowing specific recognition of its phosphate, deoxyribose and uracil moieties. Our structure thus provides a view of a productive complex specific for cleavage of uracil from DNA and also reveals the basis for the enzyme-assisted nucleotide flipping by this critical DNA-repair enzyme.

Purification and properties of the uracil DNA glycosylase from Bloom's syndrome

Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different K m, V max and were strikingly insensitive to 5-fluoracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10–100-fold higher concentrations of each analogue to achieve comparable of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.

Overproduction and characterization of the uracil-DNA glycosylase inhibitor of bacteriophage PBS2

A plasmid expression vector (pZWtacl) was constructed which allowed inducible overexpression of the uracil-DNA glycosylase (Ung) inhibitor (Ugi)-encoding gene ( ugi) in Escherichia coli. In this plasmid, the ugi gene was under the control of both its own promoter and the tac promoter. Constitutive expression of the ugi was observed in the absence of isopropyl-β- d-thiogalactopyranoside (IPTG). In the presence of 1 mM IPTG, the Ugi protein was overproduced to an approx. 16-fold higher level, and accounted for approx. 19% of the total soluble cellular proteins. Following high-level production in E. coli, the Ugi protein was purified to apparent homogeneity. Using E. coli Ung, we observed that Ugi inactivated the enzyme in a noncompetitive manner. Kinetic studies revealed a K i value (0.14 μM) of approx. twelve-fold lower than K m value (1.7 μM) of glycosylase. Ugi did not act synergistically with free uracil to inhibit E. coli Ung suggesting that uracil and Ugi could share a similar mode of inhibition.

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.

Purification of the human O6-methylguanine-DNA methyltransferase and uracil-DNA glycosylase, the latter to apparent homogeneity.

Uracil-DNA glycosylase, the enzyme that catalyzes the release of free uracil from single-stranded and double-stranded DNA, has been purified 26,600-fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil-DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme. This report also presents a 700-fold purification of HeLa S3 cell O6-methylguanine-DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.

Protein and 28S ribosomal RNA fractional turnover rates in the rat heart after abdominal aortic stenosis

The rate of synthesis of myocardial proteins and ribosomal ribonucleic acid (rRNA) was measured during the development of cardiac hypertrophy in rats using a continuous intracardiac infusion of 14C-tyrosine and 3H-uridine in unanaesthetised animals. Cardiac overload was induced by abdominal aortic stenosis. Left ventricular weight and total myocardial RNA concentration were significantly increased on day 4 after aortic stenosis (+19% and +18% respectively). On day 8 left ventricular weight reached +52% whereas RNA concentration had not increased further (+13%). The fractional turnover rates were calculated using the specific activities of intracellular free tyrosine and free uracil nucleotides (precursors) and those of protein bound tyrosine and 28S rRNA bound uridine monophosphate (products) respectively. The fractional rate of synthesis of proteins and rRNA (expressed as percentage per day) increased from 24% to 45% for proteins and from 25% to 34% for rRNA and peaked by day 2. The RNA activity, expressed as gram of protein synthesised per day and per gram of total RNA, was unchanged on day 1 and reached a maximal value on day 2 (+107%). These results suggest that the pre-existing ribosomal RNA could be underutilized under control conditions and that the boosting of RNA transcription, associated with that of protein translation, is a complementary process rather than a prerequisite for the transition period leading to hypertrophy.