UVR-induced G-C to C-G transversions from oxidative DNA damage

Many oxidizing agents induce G-C to T-A and G-C to C-G transversions, and the frequency largely depends on the oxidative conditions. Guanine is the most oxidizable base among natural bases. The typical oxidative lesion product 8-oxoguanine (8-oxoG) is responsible for G-C to T-A transversion but not for G-C to C-G transversion, and 8-oxoG is more readily oxidized than guanine because of its lowered ionization potential. Recently, imidazolone (Iz), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) have been demonstrated as oxidative lesion products of guanine and 8-oxoG, which could be responsible for G-C to C-G transversions by forming specific base pair formations.     

Molecular dynamics simulation of 7, 8-dihydro-8-oxoguanine DNA

To elucidate the effect of guanine lesion produced by the oxidative damage on DNA, 1 nanosecond molecular dynamics simulations of native and oxidized DNA were performed. The target DNA molecules are dodecamer duplex d(CGCGAATTCGCG)(2) and its derivative duplex d(C(1)G(2)C(3)(8-oxoG)(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12).d(C(13)G(14)C(15)G(16)A(17)A(18)T(19)T(20)C(21)G(22)C(23)G(24), which has one oxidized guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG), at the fourth position. The local structural change due to the lesion of 8-oxoG and the global dynamic structure of the 8-oxoG DNA were studied. It was found that the 8-oxoG DNA remained structurally stable during the simulation due to newly produced hydrogen bonds around the (8-oxoG)(4) residue. However, there were distinguishable differences in structural parameters and dynamic property in the 8-oxoG DNA. The conformation around the (8-oxoG)(4) residue departed from the usual conformation of native DNA and took an unique conformation of epsilon-zeta in B(II) conformation and chi in high anti orientation at the (8-oxoG)(4) residue, and adopted a very low helical twist angle at the C(3):G(22)-(8-oxoG)(4):C21) step. Further analysis by principal component analysis indicated that the formation of the hydrogen bonds around the (8-oxoG)(4) residue plays a role as a trigger for the conformational transition of the 8-oxoG DNA in the conformational space.     

Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.     

Product inhibition and magnesium modulate the dual reaction mode of hOgg1

8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.     

Efficient and high fidelity incorporation of dCTP opposite 7,8-dihydro-8-oxodeoxyguanosine by Sulfolobus solfataricus DNA polymerase Dpo4

DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed >/=95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.     

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae

The biological consequences of clusters containing a single strand break and base lesion(s) remain largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) in Escherichia coli. The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG on the same strand were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.     

The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool

Mismatch repair (MMR) corrects replication errors. It requires the MSH2, MSH6, MLH1, and PMS2 proteins which comprise the MutSalpha and MutLalpha heterodimers. Inactivation of MSH2 or MLH1 in human tumors greatly increases spontaneous mutation rates. Oxidation produces many detrimental DNA alterations against which cells deploy multiple protective strategies. The Ogg-1 DNA glycosylase initiates base excision repair (BER) of 8-oxoguanine (8-oxoG) from 8-oxoG:C pairs. The Myh DNA glycosylase removes mismatched adenines incorporated opposite 8-oxoG during replication. Subsequent BER generates 8-oxoG:C pairs, a substrate for excision by Ogg-1. MTH1-an 8-oxodGTPase which eliminates 8-oxodGTP from the dNTP pool-affords additional protection by minimizing 8-oxodGMP incorporation during replication. Here we show that the dNTP pool is, nevertheless, an important source of DNA 8-oxoG and that MMR provides supplementary protection by excising incorporated 8-oxodGMP. Incorporated 8-oxodGMP contributes significantly to the mutator phenotype of MMR-deficient cells. Thus, although BER of 8-oxoG is independent of Msh2, both steady-state and H(2)O(2)-induced DNA 8-oxoG levels are higher in Msh2-defective cells than in their repair-proficient counterparts. Increased expression of MTH1 in MMR-defective cells significantly reduces steady-state and H(2)O(2)-induced DNA 8-oxoG levels. This reduction dramatically diminishes the spontaneous mutation rate of Msh2(-/-) MEFs.     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein).

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

The high binding affinity of human ribosomal protein S3 to 7,8-dihydro-8-oxoguanine is abrogated by a single amino acid change

Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.     

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.     

Human polynucleotide phosphorylase reduces oxidative RNA damage and protects HeLa cell against oxidative stress

We examined HeLa cell viability and RNA oxidative damage in response to hydrogen peroxide (H₂O₂) treatment. The level of damaged RNA, measured by the content of 8-hydroxyguanosine (7,8-dihydro-8-oxoguanosine, 8-oxoG), increases depending on H₂O₂ dosage and is inversely correlated with cell viability. The elevated level of 8-oxoG in RNA decreases after removal of oxidative challenge, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. Human polynucleotide phosphorylase (hPNPase), an exoribonuclease primarily located in mitochondria, has been previously shown to bind 8-oxoG-RNA with high affinity. The role of hPNPase in HeLa cell under oxidative stress conditions is examined here. Overexpression of hPNPase reduces RNA oxidation and increases cell viability against H₂O₂ insult. Conversely, hPNPase knockdown decreases viability and increases 8-oxoG level in HeLa cell exposed to H₂O₂. Our results suggest that hPNPase plays an important role in protecting cells and limiting damaged RNA under oxidative stress.     

Ionic strength and magnesium affect the specificity of Escherichia coli and human 8-oxoguanine-DNA glycosylases

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often directs the misincorporation of dAMP during replication. To prevent mutations, cells possess an enzymatic system for the removal of 8-oxoG. A key element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria, OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but not from 8-oxoG:A. We investigated the influence of various factors, including ionic strength, the presence of Mg(2+) and organic anions, polyamides, crowding agents and two small heterocyclic compounds (biotin and caffeine) on the activity and opposite-base specificity of Escherichia coli Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A decreased sharply with increasing salt and Mg(2+) concentration, whereas the activity on 8-oxoG:C was much more stable, resulting in higher opposite-base specificity when salt and Mg(2+) were at near-physiological concentrations. This tendency was observed with both Cl(-) and glutamate as the major anions in the reaction mixture. Kinetic and binding parameters for the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were determined under several different conditions. Polyamines, crowding agents, biotin and caffeine affected the activity and specificity of Fpg or OGG1 only marginally. We conclude that, in the intracellular environment, the specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due to high ionic strength and Mg(2+).     

Age-dependent changes in 8-oxoguanine-DNA glycosylase activity are modulated by adaptive responses to physical exercise in human skeletal muscle

8-Oxo-7,8-dihydroguanine (8-oxoG) accumulates in the genome over time and is believed to contribute to the development of aging characteristics of skeletal muscle and various aging-related diseases. Here, we show a significantly increased level of intrahelical 8-oxoG and 8-oxoguanine-DNA glycosylase (OGG1) expression in aged human skeletal muscle compared to that of young individuals. In response to exercise, the 8-oxoG level was lastingly elevated in sedentary young and old subjects, but returned rapidly to preexercise levels in the DNA of physically active individuals independent of age. 8-OxoG levels in DNA were inversely correlated with the abundance of acetylated OGG1 (Ac-OGG1), but not with total OGG1, apurinic/apyrimidinic endonuclease 1 (APE1), or Ac-APE1. The actual Ac-OGG1 level was linked to exercise-induced oxidative stress, as shown by changes in lipid peroxide levels and expression of Cu,Zn-SOD, Mn-SOD, and SIRT3, as well as the balance between acetyltransferase p300/CBP and deacetylase SIRT1, but not SIRT6 expression. Together these data suggest that that acetylated form of OGG1, and not OGG1 itself, correlates inversely with the 8-oxoG level in the DNA of human skeletal muscle, and the Ac-OGG1 level is dependent on adaptive cellular responses to physical activity, but is age independent.     

Molecular Dynamics Simulation of 7, 8-dihydro-8-Oxoguanine DNA

Abstract

To elucidate the effect of guanine lesion produced by the oxidative damage on DNA, 1 nanosecond molecular dynamics simulations of native and oxidized DNA were performed. The target DNA molecules are dodecamer duplex d(CGCGAATTCGCG)2 and its derivative duplex d(C₁G₂C₃(8-oxoG)₄A₅A₆T₇T₈C₉G₁₀C₁₁G₁₂)·d(C₁₃G₁₄C₁₅G₁₆A₁₇A₁₈T₁₉T₂₀C21_G22C₂₃G₂₄), which has one oxidized guanine, 7, 8-dihydro-8-oxoguanine (8-oxoG), at the fourth position. The local structural change due to the lesion of 8-oxoG and the global dynamic structure of the 8-oxoG DNA were studied. It was found that the 8-oxoG DNA remained structurally stable during the simulation due to newly produced hydrogen bonds around the (8-oxoG)₄ residue. However, there were distinguishable differences in structural parameters and dynamic property in the 8-oxoG DNA. The conformation around the (8- oxoG)₄ residue departed from the usual conformation of native DNA and took an unique conformation of ?-ζ in B_II conformation and χ in high anti orientation at the (8-oxoG)₄ residue, and adopted a very low helical twist angle at the C₃:G₂₂—(8-oxoG)₄:C₂₁ step. Further analysis by principal component analysis indicated that the formation of the hydrogen bonds around the (8-oxoG)₄ residue plays a role as a trigger for the conformational transition of the 8-oxoG DNA in the conformational space.     

Hydrogen bonding of 7,8-dihydro-8-oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus solfataricus DNA polymerase Dpo4

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) has been shown to catalyze bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) in a highly efficient and relatively accurate manner. Crystal structures have revealed a potential role for Arg(332) in stabilizing the anti conformation of the 8-oxoG template base by means of a hydrogen bond or ion-dipole pair, which results in an increased enzymatic efficiency for dCTP insertion and makes formation of a Hoogsteen pair between 8-oxoG and dATP less favorable. Site-directed mutagenesis was used to replace Arg(332) with Ala, Glu, Leu, or His in order to probe the importance of Arg(332) in accurate and efficient bypass of 8-oxoG. The double mutant Ala(331)Ala(332) was also prepared to address the contribution of Arg(331). Transientstate kinetic results suggest that Glu(332) retains fidelity against bypass of 8-oxoG that is similar to wild type Dpo4, a result that was confirmed by tandem mass spectrometric analysis of full-length extension products. A crystal structure of the Dpo4 Glu(332) mutant and 8-oxoG:C pair revealed water-mediated hydrogen bonds between Glu(332) and the O-8 atom of 8-oxoG. The space normally occupied by Arg(332) side chain is empty in the crystal structures of the Ala(332) mutant. Two other crystal structures show that a Hoogsteen base pair is formed between 8-oxoG and A in the active site of both Glu(332) and Ala(332) mutants. These results support the view that a bond between Arg(332) and 8-oxoG plays a role in determining the fidelity and efficiency of Dpo4-catalyzed bypass of the lesion.     

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.     

Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta

Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.     

Is the repair of oxidative DNA base modifications inducible by a preceding DNA damage induction?

In mammalian cells, 7,8-dihydro-8-oxoguanine (8-oxoG) and some other oxidative guanine modifications are removed from the DNA by base excision repair, which is initiated by OGG1 protein. We have tested whether this repair is inducible in mouse embryonic fibroblasts (MEFs), MCF-7 breast cancer cells and primary human fibroblasts by a pretreatment with the photosensitizer Ro19-8022 plus light, which generates predominantly 8-oxoG, or with methyl methanesulfonate (MMS), which generates alkylated bases and abasic sites (AP sites). The results indicate that the repair rate of the oxidative guanine modifications induced by the photosensitizer was not increased if a priming dose of the oxidative or alkylating agent was applied 6 or 18h prior to a challenging dose, although pretreatments with both agents resulted in two-fold elevated glutathione levels as an indication for an adaptive response. Similarly, the activity of total protein extracts of the cells to incise at a single 8-oxoG residue in an oligonucleotide was unchanged. It has to be concluded that the repair of 8-oxoG is not inducible by oxidative or alkylation damage.     

Defective human MutY phosphorylation exists in colorectal cancer cell lines with wild-type MutY alleles

Oxidative DNA damage can generate a variety of cytotoxic DNA lesions such as 8-oxoguanine (8-oxoG), which is one of the most mutagenic bases formed from oxidation of genomic DNA because 8-oxoG can readily mispair with either cytosine or adenine. If unrepaired, further replication of A.8-oxoG mispairs results in C:G to A:T transversions, a form of genomic instability. We reported previously that repair of A.8-oxoG mispairs was defective and that 8-oxoG levels were elevated in several microsatellite stable human colorectal cancer cell lines lacking MutY mutations (human MutY homolog gene, hmyh, MYH MutY homolog protein). In this report, we provide biochemical evidence that the defective repair of A.8-oxoG may be due, at least in part, to defective phosphorylation of the MutY protein in these cell lines. In MutY-defective cell extracts, but not extracts with functional MutY, A.8-oxoG repair was increased by incubation with protein kinases A and C (PKA and PKC) and caesin kinase II. Treatment of these defective cells, but not cells with functional MutY, with phorbol-12-myristate-13-acetate also increased the cellular A.8-oxoG repair activity and decreased the elevated 8-oxoG levels. We show that MutY is serine-phosphorylated in vitro by the action of PKC and in the MutY-defective cells by phorbol-12-myristate-13-acetate but that MutY is already phosphorylated at baseline in proficient cell lines. Finally, using antibody-isolated MutY protein, we show that MutY can be directly phosphorylated by PKC that directly increases the level of MutY catalyzed A.8-oxoG repair.