8-hydroxyguanine and sodium-borohydride

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases β, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.

Topics: Bacteriophage T4; Borohydrides; DNA; DNA Adducts; DNA Replication; DNA-Directed DNA Polymerase; DNA-Formamidopyrimidine Glycosylase; DNA, Single-Stranded; Escherichia coli; Escherichia coli Proteins; Guanine; Humans; Oligonucleotides; Pyrococcus furiosus; Sulfolobus solfataricus; Transcription Termination, Genetic

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG. C-containing duplex oligo with a very low apparent k(cat) of 0.1 min(-1) at 37 degrees C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis-Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased approximately 5-fold and Michaelis-Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP.C pair (K:(d) approximately 2.8 nM) was substantially higher than for its substrate 8-oxoG.C pair (K:(d) approximately 23. 4 nM) and the affinity for its final ss-elimination product was much lower (K:(d) approximately 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.

Topics: Aminopeptidases; Bacterial Proteins; Borohydrides; Carbon-Oxygen Lyases; Cytosine; Deoxyribonuclease IV (Phage T4-Induced); DNA Adducts; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Guanine; Humans; Kinetics; Mutation; N-Glycosyl Hydrolases; Saccharomyces cerevisiae Proteins; Substrate Specificity

The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a beta-elimination reaction that cleaves phosphodiester bonds 3' and adjacent to an AP lesion in DNA. In certain situations, this is followed by a delta-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5'- and 3'-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a beta-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a delta-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the delta-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophila wild-type S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Borohydrides; Catalysis; DNA; DNA Damage; DNA Primers; DNA Repair; Drosophila; Guanine; Humans; Mutagenesis, Site-Directed; Mutagens; Ribosomal Proteins; Sequence Homology, Amino Acid

In Escherichia coli, endonuclease III (endo III) repairs the oxidation products of 8-OHGua. However, the corresponding repair enzymes in eukaryotes have not been identified. Here we report that 8-hydroxyguanine (8-OHGua) is highly sensitive to further oxidation. We also show that Ntg2, a functional homolog of endo III in Saccharomyces cerevisiae, is capable of nicking the irradiated duplex DNA containing 8-OHGua. Moreover, Ntg2 formed a stable complex with the DNA upon incubation with NaBH(4). In contrast, Ntg1, another functional homolog of endo III, showed no such activities. These findings indicate that Ntg2 is, at least in part, responsible for repairing the oxidation products of 8-OHGua in eukaryotic cells.

Topics: Borohydrides; Deoxyadenosines; Deoxycytidine; Deoxyguanosine; DNA; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Formamidopyrimidine Glycosylase; Dose-Response Relationship, Radiation; Escherichia coli Proteins; Gamma Rays; Guanine; Hydrogen Peroxide; Hydroxyl Radical; Iron; Macromolecular Substances; N-Glycosyl Hydrolases; Oxidation-Reduction; Piperidines; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Thymidine

DNA damage caused indirectly via reactive oxygen species generated during reductive activation of mitomycin C was evaluated. This oxidative DNA damage was measured by determining the formation of 8-hydroxyguanine in DNA exposed to chemically or enzymatically activated mitomycin C. The level of 8-hydroxyguanine was measured indirectly by determining formamidopyrimidine-DNA glycosylase-sensitive sites induced in plasmid DNA exposed to mitomycin C and directly by a 32P-postlabeling assay for the modified base. Activation of mitomycin C by sodium borohydride in air, by H2/Pt, or xanthine oxidase in N2 caused increases in the level of 8-hydroxyguanine. The extent of the increase varied according to the incubation conditions with the greatest increase being observed in DNA exposed to mitomycin C activated under hypoxic conditions. These results support a possible indirect mechanism for DNA damage caused by mitomycin C that is mediated by reactive oxygen species.

Topics: Anaerobiosis; Borohydrides; DNA; DNA Damage; DNA-Formamidopyrimidine Glycosylase; Guanine; Mitomycin; N-Glycosyl Hydrolases; Phosphorus Radioisotopes; Plasmids; Substrate Specificity; Xanthine Oxidase