Yoke W. Kow

Characterization of Escherichia coli Endonuclease VIII

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, β-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255–1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by β,δ-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5′-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 179, 3773–3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nm. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3′ to the reduced AP site.

Interaction of Deoxyinosine 3′-Endonuclease from Escherichia coli with DNA Containing Deoxyinosine

By using a band mobility shift assay, deoxyinosine 3′-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes. Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, K = 4 × 10M) contained one molecule of deoxyinosine 3′-endonuclease, while the slower migrating complex (complex II, K = 4 × 10M) contained two molecules of the protein bound to every molecule of duplex DNA. The endonucleolytic activity of deoxyinosine 3′-endonuclease paralleled the formation of the complex I. Interestingly, deoxyinosine 3′-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction. The formation of a stable complex required the presence of a duplex structure 5′ to the deoxyinosine residue. DNase I footprinting revealed that deoxyinosine 3′-endonuclease protected 4-5 nucleotides 5′ to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3′ to deoxyinosine were protected. Based on these results, a model is proposed for the interaction of deoxyinosine 3′-endonuclease with DNA containing deoxyinosine.

Methylperoxyl radicals as intermediates in the damage to DNA irradiated in aqueous dimethyl sulfoxide with gamma rays.

Using agarose gel electrophoresis, we have measured the yields of DNA single-strand breaks (SSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. Incubation after irradiation with the base damage repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III (endo III) results in an increase in the yield of SSBs. In the absence of dimethyl sulfoxide (DMSO) during irradiation, this increase is consistent with the yields of known substrates for FPG and endo III as determined by gas chromatography/mass spectrometry. After irradiation in the presence of 1 mol dm-3 DMSO, the increase in the yield of SSBs after enzyme incubation was further enhanced by a factor of about 5 to 7. The magnitude of this effect, the inability of acrylamide or oxygen to suppress it, and its attenuation by N,N,N,N-tetramethylphenylenediamine (TMPD) or glycerol all suggest that the methylperoxyl radical (derived from DMSO) is involved as an intermediate. Reactions of the methylperoxyl radical (or some other species derived from it) do not result in strand break damage, but are responsible for DNA base damages which are recognized by FPG and endo III.

Antibodies to oxidative DNA damage : Characterization of antibodies to 8-oxopurines

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 µmol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 µmol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 µmol/L. Both antibodies could easily detect the corresponding damage in x-irradiated fl DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10 000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.

Properties of a monoclonal antibody for the detection of abasic sites, a common DNA lesion

The abasic site is one of the most frequent changes occurring in DNA and has been shown to be lethal and mutagenic. An abasic site in DNA can be tagged by reaction with O-4-nitrobenzylhydroxylamine (NBHA), resulting in the formation of an oxime linkage between the abasic site and the NBHA moiety. In order to measure NBHA-tagged abasic sites, a monoclonal antibody was elicited against a 5-phosphodeoxyribosyl O-4-nitrobenzyl hydroxylamine-BSA conjugate. The antibody was specific for the NBHA residue as demonstrated by hapten inhibition, with IC50 values for 5-phosphodeoxyribosyl-NBHA, deoxyribosyl-NBHA, ribosyl-NBHA and NBHA of 0.3 microM, 5 microM, 5 microM and 7 microM, respectively. Other haptens examined, including benzylhydroxylamine, 5-phosphodeoxyribosyl-, deoxyribosyl-, and ribosyl-benzylhydroxylamine, showed no inhibition even at 1 mM. The antibody showed high specificity for NBHA-modified AP sites in DNA and exhibited no cross reactivity with normal DNA bases, otherwise-modified DNA bases or unmodified AP sites. Using a direct ELISA assay, the antibody detected 1 AP site (after NBHA-modification) per 10,000 base-pairs or approximately 10 femtomoles of AP sites in DNA. DNA lesions were detectable in 60Co gamma-irradiated DNA at a dose as low as 10 rad (0.1 Gy) and the production of antibody detectable sites was proportional to the gamma-ray dose. Since NBHA reacts with lesions containing an aldehyde group, the simplicity and sensitivity of the antibody assay should provide a useful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.

Deoxyinosine 3' endonuclease, a novel deoxyinosine-specific endonuclease from Escherichia coli.

Deoxyinosine in DNA arises from deamination of deoxyadenosine or misincorporation of dITP during replication. Deamination can be spontaneous or caused by nitrous acid or ionizing radiation. Deoxyinosine in DNA is potentially mutagenic, resulting in A to G transitions.* One pathway for the repair of deoxyinosine may involve hypoxanthine DNA glycosylase, an enzyme that has been partially p ~ r i f i e d . ~ . ~ But little is known about the mechanism of its action. In an effort to purify this enzyme, we isolated a novel deoxyinosine-specific endonuclease, deoxyinosine 3 endonuclease. Deoxyinosine 3 endonuclease was purified from E. coli strain BW 434 (nth-xth-). Deoxyinosine-containing bacteriophage PM2 DNA, prepared by nick translation of dITP in place of dGTP, was used as a substrate for detection of deoxyinosine-specific endonuclease activity by the alkaline fluorometric assay of Kowal~ki .~ Cell paste was first homogenized by Braun MSK homogenizer. After removal of nucleic acids by PEG precipitation, the enzyme was purified by chromatography on S Sepharose Fast Flow, followed by FPLC on Mono S, Mono Q, and Phenyl Superose. At the last step of purification, the preparation represented 24,800fold purification over the crude extract, with a 43% yield of activity. Silver-stained SDS-PAGE revealed a major polypeptide of 26 kDa with two minor contaminants. We eluted the 26-kDa polypeptide from SDS-PAGE and renatured and recovered the deoxyinosine-specific activity. Activity gel electrophoresis was also used to confirm that the deoxyinosine-specific endonuclease migrated as a 26-kDa polypeptide. The enzyme has an obligatory requirement for Mg2+ at a concentration of 50 pM. Fifty pM of CoCl, or 10 pM MnCl, could partially replace the requirement of Mgz+. The optimal pH for the enzyme is 7.5. The enzyme was found to be active on oligonucleotides containing I/T, I/C, I/A, and I/G pairs. It is also active on single-stranded DNA containing deoxyinosine, albeit at a lower rate. Deoxyinosine 3 endonuclease was also found to be active on DNA containing abasic site (AP) or urea. It cleaves the strand containing deoxyinosine or the AP site, but not the complementary strand. It does not cleave oligonucleotides with A/T pairs. It was revealed by 3 and 5 end labeling of the oligonucleotide that the enzyme cuts the second phosphodiester bond 3 to deoxyinosine. This was true for all deoxyinosine pairs and deoxyinosine-containing single-stranded DNA, as well as the AP-containing oligonucleotide. Deoxyinosine 3 endonuclease creates a nick with 3 hydroxyl group since the product of the enzyme is a substrate for nick translation. The enzyme is different from hypoxanthine DNA glycosylase and displays a novel AF endonuclease activity different from those of class I and class I1 AP endonucleases.

Base Excision Repair in E. Coli—an Overview

Damages in DNA can arise spontaneously or through the action of environmental agents. Oxidants, redox chemicals, as well as ionizing and UV irradiation can produce not only base damages, but also damages to the sugar moiety, generating abasic sites and DNA chain scissions. In E. coli, several pathways exist to facilitate the removal of these lesions. In general, oxidative base damages are repaired by the base excision repair pathway, by which damaged or modified DNA bases are recognized and removed by DNA N-glyco~ylases.~~~ These enzymes hydrolyze the N-glycosylic bond between the modified base and the sugar moiety, leaving behind an abasic (AP) site in DNA. AP sites are then further processed by AP endonucleases, which make incisions either 5 (class I1 or AP nucleotidyl hydrolases) or 3 (class I or AP lyases) to the AP sites.*v3 Class I1 AP endonucleases generate termini containing 3 hydroxyl and 5 sugar phosphate moieties at the nick sites! The 5 sugar phosphate moiety might require the action of a 5-phosphodeoxyribosyl phosphodiestera~e~ to remove the 5 sugar phosphate, generating a single-base gap. In contrast, class I AP lyases generate either a 3 4-hydroxy-2-pentenal (p-elirninati~n~) or a 3 phosphate moiety (p,b-elimination or f3-elimination plus 5 hydrolysis4). These altered 3 termini at the nick site are poor priming sites for repair polymerases such as polymerase I in E. coli; thus they require further processing by class I1 AP endonucleases. Therefore, the net result of the combined actions of N-glycosylases and AP endonucleases is the formation of a single-base gap in DNA, which can be filled in by DNA polymerase I. DNA repair is completed by a ligase reaction. As stated earlier, there are two major kinds of AP endonuclease in E. coli, true AP endonuclease (class 11) and AP lyase (class I). Class I1 AP endonuclease or 5 nucleotidyl hydrolases hydrolyze the 5 phosphodiester bond adjacent to an AP site. Exonuclease 111 and endonuclease IV are examples of class I1 AP endonuclease. They constitute more than 90% of the total cellular AP endonuclease activity in E. coli.8 These are essential AP endonucleases, since mutant cells lacking both endonuclease IV and exonuclease 111 exhibit extreme sensitivity toward agents that generate AP sites, such as methanemethyl sulfonate and x-ray? The second class of AP-recognizing activities, the class I AP endonucleases, or more correctly AP lyases, catalyze the cleavage of the phosphodiester bond 3 to an AP site by pelimination. Examples of this class of enzymes are endonucleases 111 and VIII and formamidopyrimidine N-glycosylase.24 It is interesting to note that the AP lyase activities examined so far are associated with enzymes that exhibit combined Nglycosylase/AP endonuclease activity. The presence of dual activities in these enzymes could simply be due to the fact that they form covalent Schiff base intermediates with the sugar moiety that is required for p-elimination. Much is known about the mechanism of the AP lyase activity. For endonuclease 111, the AP lyase was

Detection of Oxidative DNA Base Damages

Antibodies to a variety of oxidized DNA bases have been generated in a number of laboratories including our own (see Table I). Most of these antibodies have been elicited using protein-conjugated haptens of interest. In general, the antibodies have reasonable affinity such that appropriate sensitivity in the various assays can be achieved. The difficulty with antibodies that recognize oxidized DNA bases is that the oxidized bases do not differ largely from their unoxidized derivatives. Thus, the specificity for detecting lesions in DNA must be high especially when one considers the low level of damaged compared to undamaged bases. Even if the sensitivity of the assay can be amplified, cross-reactivity of the antibody with the unoxidized base in DNA remains an obstacle to successful detection of low levels of the oxidized base. This particular problem is not seen as often when antibodies are elicited to the chemical adducts that have features that vary quite dramatically from the unadducted base. An additional consideration is that the lesion must be stable to the procedures used during preparation of the immunogen and during DNA denaturation. The latter is usually necessary since the antibodies often do not recognize the lesion as well in duplex DNA. Despite these shortcomings, antibodies to oxidized bases have been effectively utilized to detect these lesions in oxidized or ionizing radiation-treated DNA in vitro.