Fabs specific for 8-oxoguanine: control of DNA binding.

Free radicals produce a broad spectrum of DNA base modifications including 7,8-dihydro-8-oxoguanine (8-oxoG). Since free radicals have been implicated in many pathologies and in aging, 8-oxoG has become a benchmark for factors that influence free radical production. Fab g37 is a monoclonal antibody that was isolated by phage display in an effort to create a reagent for detecting 8-oxoG in DNA. Although this antibody exhibited a high degree of specificity for the 8-oxoG base, it did not appear to recognize 8-oxoG when present in DNA. Fab g37 was modified using HCDR1 and HCDR2 segment shuffling and light chain shuffling. Fab 166 and Fab 366 which bound to 8-oxoG in single-stranded DNA were isolated. Fab 166 binds more selectively to single-stranded oligonucleotides containing 8-oxoG versus control oligonucleotides than does Fab 366 which binds DNA with reduced dependency on 8-oxoG. Numerous other clones were also isolated and characterized that contained a spectrum of specificities for 8-oxoG and for DNA. Analysis of the primary sequences of these clones and comparison with their binding properties suggested the importance of different complementarity determining regions and residues in determining the observed binding phenotypes. Subsequent chain shuffling experiments demonstrated that mutation of SerH53 to ArgH53 in the Fab g37 heavy chain slightly decreased the Fab's affinity for 8-oxoG but significantly improved its binding to DNA in an 8-oxoG-dependent manner. The light chain shuffling experiments also demonstrated that numerous promiscuous light chains could enhance DNA binding when paired with either the Fab g37 or Fab 166 heavy chains; however, only the Fab 166 light chain did so in an additive manner when combined with the Fab 166 heavy chain that contains ArgH53. A three-point model for Fab 166 binding to oligonucleotides containing 8-oxoG is proposed. We describe a successful attempt to generate a desired antibody specificity, which was not present in the animal's original immune response.

Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine.

A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo-). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate.

Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII.

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.

Uracil glycol deoxynucleoside triphosphate is a better substrate for DNA polymerase I Klenow fragment than thymine glycol deoxynucleoside triphosphate.

A major stable oxidation product of DNA cytosine is 5,6-dihydroxy-5, 6-dihydrouracil (Ug). Ug can be formed directly in DNA or in the cellular nucleotide pools by deamination of the unstable primary product, cytosine glycol. Here, we synthesized dUgTP and showed that dUgTP was incorporated in place of dTTP and was a much better substrate for the model enzyme DNA polymerase I Klenow fragment lacking proofreading activity, Kf (exo-), than deoxythymidine glycol triphosphate (dTgTP). The relative efficiency for dUgTP insertion opposite A was 10 times higher than for dTgTP; however, the extension of a primer with 3' dUg was about 100 times more efficient than the extension of a primer with 3' dTg. At the insertion step, the differences in Vmax appeared to be responsible since the apparent Kms for dUgTP and dTgTP were about the same. In contrast, both the apparent Km and Vmax for elongation of dUg were markedly different from those of dTg. Molecular modeling was performed with both Tg and Ug and provides a rational structural explanation for these observations.

A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E. coli and T4.

Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.

The phosphodiester bond 3' to a deoxyuridine residue is crucial for substrate binding for uracil DNA N-glycosylase.

Using the method of water-soluble carbodiimide-induced chemical ligation, four 27-member oligodeoxyribonucleotides containing a pyrophosphate internucleotide bond near or adjacent to a deoxyuridine residue were prepared. Escherichia coli uracil DNA N-glycosylase (UDG) activity was found to be sensitive to the presence of an internucleotide pyrophosphate bond in both single- and double-stranded DNA. The rate of uracil excision from single-stranded DNA containing a pyrophosphate bond adjacent to the uracil residue, either 3' or 5', was 0.01% and 0.1% of the rate of uracil removal from control DNA without a pyrophosphate bond, respectively. The rate of uracil excision from duplex DNA containing a pyrophosphate bond 3' or 5' to the uracil residue was also reduced, being 0.1% and 1% the rate of uracil removal from the corresponding duplex DNA control. Placing the pyrophosphate bond one nucleotide 5' or 3' away from the deoxyuridine in both single- and double-stranded oligodeoxyribonucleotides provided much better substrates for UDG. Kinetic measurements showed that the pyrophosphate bond placed adjacent to the deoxyuridine residue drastically reduced the affinity of UDG toward the modified DNA substrate, with the greatest effect occurring when the pyrophosphate bond was 3' adjacent to the deoxyuridine. The enzyme was able to excise a 3'-terminal uracil at the nicked site of a nicked duplex, DNA, provided that the terminal deoxyuridine was 3'-phosphorylated. The effect of the pyrophosphate bond on the substrate susceptibility of oligonucleotides containing deoxyuridine is discussed with respect to the mechanism of action of UDG.

Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.

Antibody Fabs that bind to DNA damages provide useful models for understanding DNA damage-specific protein interactions. BSA and RSA conjugates of the nucleoside and nucleotide derivatives of the oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), were used to immunize mice. RNA from the responders was isolated and used to repertoire clone and phage display Fabs that bind to these haptens. Direct binding and competitive enzyme-linked immunosorbent assay (ELISA) demonstrated that phage Fabs (Phabs) specific for 8-oxopurine-BSA conjugates and 8-oxoguanine were produced although the Phabs did not react with 8-oxopurines in DNA. Amino acid sequence comparisons among clones having different binding properties suggested that a relatively small portion of the binding surfaces defined by the complementarity determining regions (CDR) accounted for hapten binding specificity, whereas other regions appeared to stabilize hapten binding by interacting with protein or DNA epitopes. Chain shuffling between 8-oxopurine-BSA binding Fabs and a DNA binding Fab showed that the heavy chain of the DNA binder conferred DNA binding capacity to the light chain of only one of the 8-oxopurine-BSA binders. Homology modeling of the 8-oxoG-specific clone g37 showed significant similarities to two previously isolated monoclonal antibodies specific for single-stranded nucleic acids. In the 8-oxoG Fab, which did not bind to DNA, the presumptive DNA binding canyon was blocked by heavy chain residues in the CDR2 region and appeared to lack part of the canyon wall due to the different placement of the light chain framework region.

Uracil DNA N-glycosylase distributively interacts with duplex polynucleotides containing repeating units of either TGGCCAAGCU or TGGCCAAGCTTGGCCAAGCU.

Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.

5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently incorporated into DNA by exonuclease-free Klenow fragment than 8-oxopurine deoxynucleoside triphosphates.

Recent studies with 8-oxodeoxyguanosine triphosphate (8-oxodGTP) have suggested that incorporation of oxidized nucleotides from the precursor pool into DNA may have deleterious effects. Here we show that 5-hydroxydeoxycytosine triphosphate (5-OHdCTP) and 5-hydroxydeoxyuridine triphosphate (5-OHdUTP) are more efficient substrates than 8-oxodGTP for Escherichia coli DNA polymerase I Klenow fragment lacking proofreading activity, while 8-oxodeoxyadenosine triphosphate (8-oxodGTP, 5-OHdCTP can mispair with dA in DNA but with lower efficiency. Since the 5-hydroxypyrimidines are present in normal and oxidized cellular DNA in amounts similar to the 8-oxopurines, these data suggest that enzymatic mechanisms might exist for removing them from the DNA precursor pools.

New substrates for old enzymes. 5-Hydroxy-2'-deoxycytidine and 5-hydroxy-2'-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2'-deoxyuridine is a substrate for uracil DNA N-glycosylase.

5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.

Alpha-deoxyadenosine, a major anoxic radiolysis product of adenine in DNA, is a substrate for Escherichia coli endonuclease IV.

Oligonucleotides containing a unique alpha-deoxyadenosine or tetrahydrofuran (a model abasic site) were synthesized using phosphoramidite chemistry. Repair enzymes from Escherichia coli, including endonucleases III, IV, and VIII, exonuclease III, formamidopyrimidine N-glycosylase, and deoxyinosine 3'-endonuclease, as well as UV dimer N-glycosylases from T4 (den V) and Micrococcus luteus, were examined for their ability to recognize alpha-deoxyadenosine and tetrahydrofuran. In agreement with prior studies, a tetrahydrofuran-containing oligonucleotide was a substrate for endonuclease IV and exonuclease III, but not for the other repair enzymes. However, an oligonucleotide containing alpha-deoxyadenine was a substrate only for endonuclease IV. Competitive inhibition studies with both substrates confirmed that the activity recognizing alpha-deoxyadenine was endonuclease IV and not a possible contaminant in the endonuclease IV preparation. Using E. coli extracts, the activity that recognized alpha-deoxyadenine was dependent on nfo, the structural gene of endonuclease IV, further substantiating that endonuclease IV is the enzyme that recognized alpha-deoxyadenine. Kinetic measurements indicated that alpha-deoxyadenosine was as good a substrate for endonuclease IV as tetrahydrofuran; the Km and Vmax values for both substrates were similar. Using substrates that were labeled at either the 3'- or 5'-terminus, endonuclease IV was shown to hydrolyze the phosphodiester bond 5' to either alpha-deoxyadenosine or tetrahydrofuran, leaving the lesion, alpha-deoxyadenosine or tetrahydrofuran, on the 5'-terminus of the nicked site. The ability of endonuclease IV to recognize alpha-deoxyadenosine suggests that endonuclease IV is able to recognize a new class of DNA base lesions that is not recognized by other DNA N-glycosylases and AP endonucleases.

Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.

A novel method for site specific introduction of single model oxidative DNA lesions into oligodeoxyribonucleotides.

Calf thymus terminal deoxynucleotidyl transferase was used to incorporate several products of oxidative base damage onto the 3' end of oligodeoxyribonucleotides. Under the defined conditions described in this report, single residues of dihydrothymine, beta-ureidoisobutyric acid, thymine glycol, urea, 7-hydro-8-oxoadenine, 7-hydro-8-oxoguanine, 5-hydroxycytosine and 5-hydroxyuracil were incorporated into oligodeoxyribonucleotides of different lengths. The reaction is both efficient and cost effective. The 3' termini of the reaction products were suitable substrates for ligation by phage T4 DNA ligase, facilitating greatly the construction of oligodeoxyribonucleotides containing unique and site specific oxidative DNA lesions.

A new affinity reagent for the site-specific, covalent attachment of DNA to active-site nucleophiles: application to the EcoRI and RsrI restriction and modification enzymes.

A modified oligodeoxyribonucleotide duplex containing an unnatural internucleotide trisubstituted 3' to 5' pyrophosphate bond in one strand [5'(oligo1)3'-P(OCH3)P-5'(oligo2) 3'] reacts with nucleophiles in aqueous media by acting as a phosphorylating affinity reagent. When interacted with a protein, a portion of the oligonucleotide [--P-5'(oligo2)3'] becomes attached to an amino acid nucleophilic group through a phosphate of the O-methyl-modified pyrophosphate linkage. We demonstrate the affinity labeling of nucleophilic groups at the active sites of the EcoRI and RsrI restriction and modification enzymes with an oligodeoxyribonucleotide duplex containing a modified scissile bond in the EcoRI recognition site. With the EcoRI and RsrI endonucleases in molar excess approximately 1% of the oligonucleotide becomes attached to the protein, and with the companion methyltransferases the yield approaches 40% for the EcoRI enzyme and 30% for the RsrI methyltransferase. Crosslinking proceeds only upon formation of a sequence-specific enzyme-DNA complex, and generates a covalent bond between the 3'-phosphate of the modified pyrophosphate in the substrate and a nucleophilic group at the active site of the enzyme. The reaction results in the elimination of an oligodeoxyribonucleotide remnant that contains the 3'-O-methylphosphate [5'(oligo1)3'-P(OCH3)] derived from the modified phosphate of the pyrophosphate linkage. Hydrolysis properties of the covalent protein-DNA adducts indicate that phosphoamide (P-N) bonds are formed with the EcoRI endonuclease and methyltransferase.

[Study of the mutagenic properties of oligonucleotides containing and internucleotide pyrophosphate bond using a mutation system based on phage M13]. / Issledovanie mutagennykh svoistv oligonukleotidov, soderzhashchikh mezhnukleotidnoe pirofosfatnoe zveno, c pomoshch'iu mutatsionnoi sistemy na osnove faga M13.

Mutagenic properties of oligonucleotides with pyrophosphate internucleotide bond was studied. It was shown that the pyrophosphate bond in the oligo structure does not induce mutations but promotes a more efficient induction of marker deletions predetermined by the nucleotide sequence as compared to the native oligonucleotide. Marker deletion induction proceeds according to the repair mechanism as homozygotes dominate in the mutant generation.

[Hybridase cleavage of RNA. I. Oligonucleotide probes for regiospecific RNA cleavage]. / Gibridaznoe rasshcheplenie RNK. I. Oligonukleotidnye zondy dlia regiospetsificheskogo rasshchepleniia RNK.

New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.

[Introduction of tri- and tetraphosphate internucleotide bonds into oligodeoxyribonucleotides]. / Vvedenie mezhnukleotidnykh tri- i tetrafosfatnykh sviazei v oligodezoksiribonukleotidy.

Dodecadeoxyribonucleotides d(AGCTTGpppGCTGCA) and d(AGCTTGppppGCTGCA) were obtained by template directed chemical condensation induced by a water-soluble carbodiimide.

[Transfer of "artificial transposons" constructed on the basis of insertion element IS1]. / Peremeshchenie "iskusstvennykh transpozonov", skonstruirovannykh na osnove insertsionnogo élementa IS1.

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.