The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.     

Syntheses and Interactions of Oligodeoxyribonucleotides Containing 2′-Amino-2′-Deoxyuridine

Abstract

Oligodeoxyribonucleotides containing 2′-amino-2′-deoxy-uridine (dU) were synthesized and their ability to form duplexes with complementary DNA or RNA oligonucleotides was studied. Substitution of dU with dU in these oligomers results in lowered Tms of the duplexes.     

Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase

Recombinant uracil-DNA glycosylase (UDG) from Aeropyrum pernix (A. pernix) was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG) were studied using oligonucleotides carrying a deoxyuracil (dU) base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.     

From footprint to toeprint: a close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA.

The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus sequence: 5'-TTA/TTNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique. (i) dU at the positions of T2, T4, T7' or T9' completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T3' on the complementary strand of teh box 5'-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position. In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Homooligomeric dA·dU and dA·dT Sequences in Parallel and Antiparallel Strand Orientation: Consequence of the 5-methyl Groups on Stability,Structure and Interaction with the Minor Groove Binding Drug HOECHST 33258

Abstract

Oligodeoxyribonucleotides containing dA·dU base combinations were shown to form parallel stranded DNA. CD spectra and hyperchromicity profiles provide evidence that the structure is very similar to that of a related parallel stranded dA·oligomer. Thermal denaturation studies show that these parallel dAdU sequences are significantly less stable than their dA·analogues in either antiparallel or parallel stranded orientations. The stabilizing effect of the 5- methyl group is similar for parallel and antiparallel sequences. The minor groove binding drug Hoechst 33258 binds with similar affinity to APS dA·and APS dA·dU sequences. However, binding to the PS dA·hairpin is significantly impaired as a consequence of the different groove dimensions and the presence of thymine methyl groups at the binding site. This results in an 8.6 kJmoF reduced free energy of binding for the PS dA·sequence. Replacement of the bulky methyl group with a hydrogen (ie. T -> U) results in significantly stronger Hoechst 33258 binding to the parallel dA·dU sequences with a penalty of only 4.1 kJmol^?1. Our data demonstrate that although Hoechst 33258 detects the altered groove, it is still able to bind a PS duplex containing dA·dU base pairs with high affinity, despite the large structural differences from its regular binding site in APS DNA.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

The Crystal Structure of d(CCCCGGGG): A New A-Form Variant with an Extended Backbone Conformation

Abstract

The crystal structure of d(CCCCGGGG) has been determined at a resolution of 2.25Å. The oligomers crystallize as A-DNA duplexes occupying crystallographic two-fold axes. The backbone conformation is, in general, similar to that observed in previously reported crystal structures of A-DNA fragments, except for the central linkage, where it adopts an extended structure resulting from all trans conformation at the P-05′-C5′-C4′ bonds. This type of conformation facilitates interstrand stacking between the guanines at the C-G site. The local helix twist at this step is very small (25°) compared to an overall average of 33.5°. The unique structure of the C-G base-pair step, namely the extended backbone and the distinct stacking geometry, may be an important feature in the recognition mechanism between double- stranded DNA molecules and restriction endonucleases such as Msp I, which cuts the sequence CCGG very specifically with a rate unaffected by neighboring base pairs.     

Oligomers Containing 2′-Deoxyinosine Isosteres as Ambiguous Nucleoside or 1,3-Propanediol as Nucleoside Substitute

Abstract

Three new appropriately protected phosphoramidites have been synthesized. Two of them (1 and 2) are isosteric to that of inosine (3) [1], one is a derivative of 1′3-propanediol (4). Whereas the inosine isosteres contain an ambiguous base recognizing adenine, guanine as well as cytosine residues in double stranded DNA-fragments the 1,3-propanediol unit can be seen as a simple nucleoside substitute in a DNA chain. It contains only those structural elements necessary to form the sugar/phosphate backbone, without supplying the DNA with either a base [21 or a 2′-deoxyribofuranosyl moiety.     

Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus ΔH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus ΔH. After incision at the 5′-side of a 2′-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3′-OH terminus and carry out repair synthesis generating a 5′-flap structure that is resolved by mthFEN, a 5′-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.     

The Conformation of Single Stranded Oligonucleotides and of Oligonucleotide-Oligopeptide Complexes from their Rotation Relaxation in the Nanosecond Time Range

Abstract

The conformation of single stranded oligonucleotides is analysed by measurements of their rotation time constants. The oligomers are aligned to some degree by short electric field pulses; after pulse termination the transition to a random orientation is followed by measurements of the linear dichroism. An efficient deconvolution procedure is developed for evaluation of the experimental data obtained in the ns-time range. The increase of rotation time constants observed for chain lengths in the range from 14 to 22 residues are interpreted according to a weakly bending rod model providing a persistence length and a Stokes' diameter. The Stokes' diameters obtained for ribo- and deoxyriboadenylates are about 13Å, in approximate agreement with the expectation for a single stranded helix. The persistence length L = 53Å corresponding to ～16 nucleotide residues found for riboadenylates at 2°C appears to reflect relatively strong stacking interactions at this temperature. However, a comparison with the average length of stacked residues evaluated from available thermodynamic parameters of base stacking indicate that unstacked residues are not completely flexible. Apparently the ribose-phosphate chain provides an essential contribution to the stiffness of oligomers and polymers, even when the bases are unstacked. Addition of 100μM Mg²⁺ leads to an increase of the persistence length to 88Å. Corresponding measurements with deoxyriboadenylates show a slightly lower value of the persistence length than that found for riboadenylates. Addition of LysTrpLys and LysTyrLys to A(pA)₁₉ leads to an increase of the rotation time constant, which corresponds approximately to a length increment by one residue per bound peptide. Since controls performed with LysLeuLys do not show any similar effect, the increase of the time constants induced by LysTrpLys and LysTyrLys is attributed to intercalation of the aromatic amino acids.     

Synthesis and Molecular Modeling of Novel HSV1 Uracil-DNA Glycosylase Inhibitors

Abstract

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported ¹. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved². Starting with the optimized model of binding between 6-(4′-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.     

5′-Phosphorylation of Oligonucleotides with Phosphorous Acid in Automated DNA Synthesis

Abstract

Coupling of phosphorous acid in automated DNA synthesis using H-phosphonate methodology leads to 5′-5′ linked dimers and 5′-H-phosphonates. The yield is dependent on the phosphorous acid concentration, chain length of the oligomer, and pore size of the support. 5′-Phosphate oligomers are obtained from the H-phosphonate oligomers by silylation and oxidation.     

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine: Introduction of a Novel Class of Phosphodiester Internucleoside Linkages

Abstract

3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine (5) was synthesized and converted into the phosphoramidite building block 8. Novel oligodeoxynucleotide analogues containing 4′-C-hydroxymethyl phosphodiester internucleoside linkages were synthesized on an automated DNA-synthesizer. The hybridization properties and enzymatic stability were studied on oligomers with one to four modifications. The 3′-end modified oligodeoxynucleotides were resistent towards 3′-exonuclease degradation and showed only moderate lowered affinity towards complementary DNA compared with oligodeoxynucleotides bearing modifications in the middle.     

Dynamic Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Evidence of Intercalator Creeping

Abstract

We have used one and two dimensional exchange ¹H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1′-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)₂ ( 1 ) and d(CGCTAGCTAGCG)₂ ( 2 ). TOTO binds preferentially to the (5′-CTAG-3′)₂ sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1 -TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2 -TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process (“creeping”) along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Evidence for the presence of chromatin-bound deoxyribonucleic acid phosphatase-exonuclease in Yoshida sarcoma ascites cells

An enzyme which cleaves the phosphoester bond of 3′-phosphoryl termini of DNA was isolated and purified from the chromatin of Yoshida sarcoma cells. The DNA phosphatase is specific for only 3′-phosphorylated DNA with a lesser activity for its single stranded form. Phosphoester bonds of various nucleotides, 3′-phosphorylated RNA and 5′-phosphorylated DNA were not hydrolysed by the enzyme. The DNA phosphatase required 10 mM MgCl₂, and was inactivated by 70 % with 1 mM ?-chloromercuribenzoate and completely by heat treatment at 70° for 5 min. Furthermore, an exonuclease activity could not be separated from the purified DNA phosphatase.