The mechanism of free base formation from DNA by bleomycin. A proposal based on site specific tritium release from Poly(dA.dU)

Poly(dA.dU), which is specifically tritiated at the 1'-, 2'- (ribo configuration), 3'-, or 4'-position of deoxyuridine, has been synthesized and the fate of the tritium has been determined upon degradation of the polymer by bleomycin, Fe(II), and O2. No tritium is labilized from the 1'-3H-labeled polymer as 3H2O; however, the resulting 3-(uridin-1'-yl)-2-propenal (uracil propenal) has the expected specific activity. The 2'-3H-labeled polymer affords 3H2O and no label in the uracil propenal. This result and the lack of solvent incorporation into the uracil propenal suggest that proton abstraction from C-2' to afford the trans-propenal is highly stereospecific. For the 3'-3H-labeled polymer, 3H2O is formed and the specific activity of the uracil propenal is identical to that of the deoxyuridine. This suggests that the labilization of the 3'-H is exclusively associated with free uracil formation. 3H2O is also formed from the 4'-3H-labeled polymer. These findings along with previous studies are consistent with the formation of uracil propenal and free uracil by the trapping of the initially formed 4'-radical species by O2 or by a monooxygen species, respectively.     

DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for C-O bond cleavage accompanying base propenal formation

Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position.     

Mechanism of bleomycin: evidence for 4'-ketone formation in poly(dA-dU) associated exclusively with free base release

Incubation of poly(dA-[3'-3H]dU), poly(dA-[5'-3H]dU), or poly(dA-[5'-3H]dT) under a variety of conditions with activated bleomycin resulted in the production of free nucleic acid base, base propenal, and a small amount of 3H2O. Adjustment of the terminated reaction mixture to pH 10 and incubation at 95 degrees C resulted in a time-dependent increase in 3H2O to an amount equal to the amount of free base. If the terminated reaction mixture was incubated with NaBH4 prior to the heat and alkaline treatment, the release of 3H2O was significantly inhibited. These results are consistent with the generation by activated bleomycin of a 4'-ketone yielding free base, with the exchange of the 3'- and 5'-hydrogens by enolization and with the alkaline-induced strand scission occurring from this intermediate.     

The DNA cleavage mechanism of iron-bleomycin. Kinetic resolution of strand scission from base propenal release

The action of iron-bleomycin and O2 in cleaving DNA has been resolved into two kinetic events following the initial attack on DNA by the kinetically competent drug species, "activated bleomycin." At 4 degrees C, DNA strand scission, monitored both viscometrically and fluorimetrically (t1/2 = 2.5-5 min), precedes the release from DNA of nucleic base propenals, which is half complete in about 40 min. Therefore, a moderately stable intermediate consisting of cleaved DNA bearing a base propenal precursor is formed. The release of tritium from deoxyribose carbon-2 occurs at the time of DNA scission, which is consistent with the base propenal precursor retaining the deoxyribose-3'-phosphate bond. Specific mechanistic proposals are discussed.     

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.     

Tritium labelling of nucleic acids accompanied by conversion of cytosine to uracil.

A new method of incorporation of tritium into nucleic acids with an accompanying conversion of cytosine to uracil is proposed. The method is based on the reaction of nucleic acids with bisulfite in the presence of 3H2O. Under certain conditions poly(C) is quantitatively converted to a radioactive poly(U), whereas similar bisulfite treatment of poly(U) does not result in any tritium incorporation. Specificity of the reaction is confirmed by the results of analysis of modified tRNA and rRNA. Incubation of tRNA with bisulfite and 3H2O does not lead to cleavage of the polynucleotide chain. Similar treatment of the denatured DNA results in tritium incorporation into DNA which is accompanied by a conversion of cytosine to uracil. There is virtually no reaction between native DNA and bisulfite. Only certain cytosone residues in yeast tRNAVal/2a interact with bisulfate providing that reaction is carried out under sufficiently mild conditions.     

Microsome-stimulated activation of ferrous bleomycin in the presence of DNA

The inhibition of Fe(II)-bleomycin activation, by a large excess of DNA, is overcome by rat liver microsomes in the presence of NADPH. This release of inhibition, as indicated by increased yields of base propenal from DNA scission, is enhanced by menadione, is inhibited by superoxide dismutase, and is therefore dependent on superoxide anion. Microsomal activation of Fe(II)-bleomycin doubles the stoichiometry of base propenal yield compared to that obtained upon self-activation of the drug; 0.5 mol of base propenal is formed and 0.5 mol of NADPH is oxidized per mol of Fe(II)-bleomycin. In the presence of a large excess of DNA, Cu(II)-bleomycin is not reduced and Fe(III)-bleomycin is neither reduced nor activated by microsomes in cases where activation of Fe(II)-bleomycin is maximal. We suggest that in vivo, electron transport enzymes at or near the nucleus can stimulate the activation of Fe(II)-bleomycin under conditions where self-activation does not readily occur.     

Analysis of products formed during bleomycin-mediated DNA degradation

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2'-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.     

Phenotypic changes, signaling pathway, and functional correlates of GPR17-expressing neural precursor cells during oligodendrocyte differentiation

The developing and mature central nervous system contains neural precursor cells expressing the proteoglycan NG2. Some of these cells continuously differentiate to myelin-forming oligodendrocytes; knowledge of the destiny of NG2(+) precursors would benefit from the characterization of new key functional players. In this respect, the G protein-coupled membrane receptor GPR17 has recently emerged as a new timer of oligodendrogliogenesis. Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway, and assess the functional consequences of GPR17 activation by its putative endogenous ligands, uracil nucleotides and cysteinyl leukotrienes (cysLTs). GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. Specifically, GPR17 decorated two subsets of slowly proliferating NG2(+) OPCs: (i) morphologically immature cells expressing other early proteins like Olig2 and PDGF receptor-α, and (ii) ramified preoligodendrocytes already expressing more mature factors, like O4 and O1. Thus, GPR17 is a new marker of these transition stages. In OPCs, GPR17 activation by either uracil nucleotides or cysLTs resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Finally, uracil nucleotides promoted and GPR17 inhibition, by either antagonists or siRNAs, impaired the normal program of OPC differentiation. These data have implications for the in vivo behavior of NG2(+) OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair.     

Role of molecular oxygen in lignin peroxidase reactions

Homogeneous lignin peroxidase (diarylpropane oxygenase) oxidized veratryl alcohol to veratryl aldehyde under anaerobic conditions in the presence of either H2O2, m-chloroperoxybenzoic acid (mCPBA), or p-nitroperoxybenzoic acid (pNPBA). Lignin peroxidase also oxidized the 1-(3',4'-diethoxyphenyl)-1,2-dihydroxy-(4"-methoxyphenyl)-propane I under anaerobic conditions in the presence of mCPBA to yield 3,4-diethoxybenzaldehyde III and 1-(4'-methoxyphenyl)-1,2-dihydroxyethane IV. In contrast to what occurs under aerobic conditions, under anaerobic conditions no 2-hydroxy-1-(4'-methoxyphenyl)-1-oxoethane V was obtained. During the diarylpropane I cleavage under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of the phenylglycol IV. Lignin peroxidase also hydroxylated 1-(4'-ethoxy-3'-methoxyphenyl)propane II at the alpha-position to yield 1-(4'-ethoxy-3'-methoxyphenyl)-1-hydroxypropane VI under anaerobic conditions in the presence of mCPBA. During the phenylpropane II hydroxylation under anaerobic conditions, 18O from H2(18)O was incorporated into the alpha-position of VI. These results are rationalized according to a mechanism involving an initial one-electron oxidation of the diarylpropane I by the lignin peroxidase compound I to form a benzene pi cation radical which undergoes alpha, beta cleavage to produce a benzaldehyde and a C6C2 benzylic radical. The latter is then attacked by O2 to form a hydroperoxy radical which may decompose through a tetroxide to form the phenylglycol IV and phenylketol V. Under anaerobic conditions the C6C2 benzylic radical is probably oxidized to a carbonium ion which would be subsequently attacked by H2O to yield the phenylglycol V.     

A new catalyst for reductive cleavage of methylated glycans

Several per-O-methylated D-glucans and D-fructans were used as models in an attempt to identify new catalysts for carrying out reductive cleavage. Included in these model studies were several D-glucans that contained 4-linked D-glucopyranosyl residues as well as one having a 4-linked D-glucitol residue, as both types of residue had previously been found to give rise to substantial proportions of artifactual products. These studies led to the development of a new catalyst for carrying out reductive cleavage, namely, a mixture of 5 equivalents of trimethylsilyl methanesulfonate (Me3SiOSO2Me) and 1 equivalent of boron trifluoride etherate (BF3 . Et2O) per equivalent of acetal. This new catalyst was found to accomplish the reductive cleavage of per-O-methylated, 4-linked D-glucopyranosyl residues and 4-linked D-glucitol residues, to give only the expected derivatives of 1,5-anhydro-D-glucitol and D-glucitol, respectively. The mixture of Me3SiOSO2Me and BF3 . Et2O also catalyzed reductive cleavage of the D-fructofuranosyl residues of per-O-methylated sucrose and inulin, to give only the expected derivatives of 2,5-anhydro-D-mannitol and 2,5-anhydro-D-glucitol. Indeed, when used alone, Me3SiOSO2Me also rapidly catalyzed the reductive cleavage of D-fructofuranosyl residues, but, under the same conditions, D-glucopyranosyl residues were unaffected. The results of these and other model studies demonstrated that catalysis of reductive cleavage by the mixture of Me3SiOSO2Me and BF3 . Et2O occurs in a synergistic manner. Examination of the mixture of Me3SiOSO2Me and BF3 . Et2O by 1H-n.m.r. spectroscopy demonstrated that a reaction occurs to generate trimethylsily fluoride and species of the type F2BOSO2Me, FB(OSO2Me)2, or B(OSO2Me)3 via ligand exchange.     

Differential role of base excision repair proteins in mediating cisplatin cytotoxicity

Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase β (Polβ) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polβ polymerase functions.     

An application of 3D-QSAR to the analysis of the sequence specificity of DNA alkylation by uracil mustard.

The sequence specificity of DNA alkylation by uracil mustard was examined using a novel three-dimensional QSAR method known as HASL, or the hypothetical active site lattice. The structures of a variety of 4-mer sequences obtained from pBR322 and SV40 were related to their degree of guanine-N7 alkylation by uracil mustard. The resulting correlations were found to point to a significant contribution from bases on the 3' side of the target guanine nucleotide. The HASL models derived from the analysis of 52 guanine-containing 4-mer sequences were used to highlight those atomic features in the favored TGCC sequence that were found most important in determining specificity. It was found that the NH2-O systems present in the two GC base pairs on the 3' side of the target guanine were significantly correlated to the degree of alkylation by uracil mustard. This finding is consistent with a prealkylation binding event occurring between these sites along the major groove and the uracil mustard O2/O4 system.     

Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding

The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.     

Analysis of linkage positions in a polysaccharide containing nonreducing, terminal alpha-D-glucopyranosyluronic groups by the reductive-cleavage method

The fate of terminal (nonreducing) alpha-D-glucopyranosyluronic groups under reductive cleavage conditions was investigated by using the Klebsiella K2 (strain NCTC-418) capsular polysaccharide. Treatment of the fully methylated polysaccharide (1) with triethylsilane and a mixture of trimethylsilyl methanesulfonate (Me3SiOSO2CH3) and boron trifluoride etherate (BF3.Et2O) as the catalyst, resulted in complete cleavage of all glycosidic linkages to yield the expected products, namely 3-O-acetyl-1,5-anhydro-2,4,6-tri-O-methyl-D-glucitol (2), 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-mannitol (3), 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-D-glucitol (4), and methyl 2,6-anhydro-3,4,5-tri-O-methyl-L-gulonate. Treatment of 1 with trimethylsilyl trifluoromethanesulfonate (Me3SiOSO2CF3) as the catalyst resulted in incomplete cleavage of the glycosidic linkage of the methylated D-glucopyranosyluronic group, to yield 4-O-acetyl-1,5-anhydro-2,6-di-O-methyl- 3-O-(methyl2,3,4-tri-O-methyl-alpha-D-glucopyranosyluronate )-D-mannitol (9). Reductive cleavage of 1 in the presence of BF3.Et2O resulted in incomplete cleavage of all glycosidic linkages and gave rise to all four dimers (including 9) that could be formed from a tetrasaccharide repeating unit. The proposed structures of these dimers are based upon their composition, as established by chemical ionization mass spectrometry and by the reported structure of the polysaccharide. A small proportion of 1,5-anhydro-2,4,6-tri-O-methyl-3-O-(methyl 2,3,4-tri-O-methyl-alpha-D-glucopyranosyluronate)-D-mannitol (12) was also detected in the products of the BF3.Et2O-catalyzed reductive cleavage. The presence of 12 is chemical evidence for the phase of the tetrasaccharide repeating unit in the polysaccharide. The reductive cleavage of 1 was also accomplished after reduction of its ester groups with lithium aluminum hydride. Complete cleavage of all glycosidic linkages was observed when either Me3SiOSO2CF3 or Me3SiOSO2CH3-BF3.Et2O was used to catalyze reductive cleavage, and anhydroalditols 2, 3, 4, and 6-O-acetyl-1,5-anhydro-2,3,4-tri-O-methyl-D-glucitol were produced, as expected.     

Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.     

Oxidative damage of DNA by chromium(V) complexes: relative importance of base versus sugar oxidation

Chromium(V)-mediated oxidative damage of deoxy-ribonucleic acids was investigated at neutral pH in aqueous solution by utilizing bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) (I) and bis(hydroxyethyl)-amino-tris(hydroxymethyl)methane)oxochromate(V) (II). Single-stranded and double-stranded (ds) calf thymus and human placenta DNA, as well as two oligomers, 5'-GATCTAGTAGGAGGACAAATAGTGTTTG-3' and 5'-GATCCAAGCAAACACTATTTGTCCTCCTACTA-3', were reacted with the chromium(V) complexes. Most products were separated and characterized by chroma-tographic and spectroscopic methods. Polyacrylamide gel electrophoresis experiments reveal more damage at G sites in comparison to other bases. Three primary oxidation products, 5-methylene-2-furanone (5-MF), furfural and 8-oxo-2'-deoxyguanosine, were characterized. A minor product, which appears to be thymine propenal, was also observed. The dsDNA produces more furfural than furanone. The formation of these two products resulted from hydrogen ion or hydride transfer from C1' and C5' positions of the ribose to the oxo-chromium(V) center. Since no enhancements of these products (except propenal) were observed in the presence of oxygen, mechanisms pertaining to the participation of activated oxygen species may be ruled out. The oxidation of the G base is most likely associated with an oxygen atom transfer from the oxo-metallates to the double bond between C8 and N7 of the purine ring. The formation of the propenal may be associated with an oxygen-activated species, since a marginal enhancement of this product was observed in the presence of oxygen. The formation of furfural in higher abundance over 5-MF for dsDNA was attributed to the ease of hydrogen ion (or hydride transfer) from the C5' compared to C1' position of the ribose within a Cr(V)-DNA intermediate in which the metal center is bound to the phosphate diester moiety.     

Repair of deaminated base damage by Schizosaccharomyces pombe thymine DNA glycosylase

Thymine DNA glycosylases (TDG) in eukaryotic organisms are known for their double-stranded glycosylase activity on guanine/uracil (G/U) base pairs. Schizosaccharomyces pombe (Spo) TDG is a member of the MUG/TDG family that belongs to a uracil DNA glycosylase superfamily. This work investigates the DNA repair activity of Spo TDG on all four deaminated bases: xanthine (X) and oxanine (O) from guanine, hypoxanthine (I) from adenine, and uracil from cytosine. Unexpectedly, Spo TDG exhibits glycosylase activity on all deaminated bases in both double-stranded and single-stranded DNA in the descending order of X > I > U  O. In comparison, human TDG only excises deaminated bases from G/U and, to a much lower extent, A/U and G/I base pairs. Amino acid substitutions in motifs 1 and 2 of Spo TDG show a significant impact on deaminated base repair activity. The overall mutational effects are characterized by a loss of glycosylase activity on oxanine in all five mutants. L157I in motif 1 and G288M in motif 2 retain xanthine DNA glycosylase (XDG) activity but reduce excision of hypoxanthine and uracil, in particular in C/I, single-stranded hypoxanthine (ss-I), A/U, and single-stranded uracil (ss-U). A proline substitution at I289 in motif 2 causes a significant reduction in XDG activity and a loss of activity on C/I, ss-I, A/U, C/U, G/U, and ss-U. S291G only retains reduced activity on T/I and G/I base pairs. S163A can still excise hypoxanthine and uracil in mismatched base pairs but loses XDG activity, making it the closest mutant, functionally, to human TDG. The relationship among amino acid substitutions, binding affinity and base recognition is discussed.     

Use of a 1-hydroxybenzotriazole activated phosphorylating reagent towards the synthesis of short RNA fragments in solution.

Evidence will be presented to show that the reagents 3a-c (X = O) obtained by the reaction of 2-chlorophenylphosphorodichloridate with 1-hydroxybenzotriazole or 1-hydroxy-6-trifluoromethyl (6-nitro) benzotriazoles, respectively, do not give, under normal coupling conditions, phosphorylation of the lactam functions in uracil or guanine. Reagent 3b (X = O) proved to be very convenient for the in situ formation of 3'-5'-phosphotriester linkage. The latter will be illustrated by the synthesis of several RNA fragments.