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An organometallic inhibitor for the human repair enzyme 7,8-dihydro-8-oxoguanosine triphosphatase.

ABSTRACT: The probe-based discovery of the first small-molecule inhibitor of the repair enzyme 8-oxo-dGTPase (MTH1) is presented, which is an unconventional cyclometalated ruthenium half-sandwich complex. The organometallic inhibitor with low-nanomolar activity displays astonishing specificity, as verified in tests with an extended panel of protein kinases and other ATP binding proteins. The binding of the organometallic inhibitor to MTH1 is investigated by protein crystallography.

SUBMITTER: Streib M

PROVIDER: S-EPMC4108727 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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An organometallic inhibitor for the human repair enzyme 7,8-dihydro-8-oxoguanosine triphosphatase.

Streib Manuel M Kräling Katja K Richter Kristin K Xie Xiulan X Steuber Holger H Meggers Eric E

Angewandte Chemie (International ed. in English) 20131120 1

The probe-based discovery of the first small-molecule inhibitor of the repair enzyme 8-oxo-dGTPase (MTH1) is presented, which is an unconventional cyclometalated ruthenium half-sandwich complex. The organometallic inhibitor with low-nanomolar activity displays astonishing specificity, as verified in tests with an extended panel of protein kinases and other ATP binding proteins. The binding of the organometallic inhibitor to MTH1 is investigated by protein crystallography. ...[more]

PMID: 24258965

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7,8-Dihydro-8-oxoguanosine Lesions Inhibit the Theophylline Aptamer or Change Its Selectivity.

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Mutagenic Replication of the Major Oxidative Adenine Lesion 7,8-Dihydro-8-oxoadenine by Human DNA Polymerases.

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Base excision repair of reactive oxygen species-initiated 7,8-dihydro-8-oxo-2'-deoxyguanosine inhibits the cytotoxicity of platinum anticancer drugs.

Project description:Anticancer therapy with cisplatin and oxaliplatin is limited by toxicity and onset of tumor resistance. Both drugs form platinum-DNA cross-linked adducts, and cisplatin causes oxidative DNA damage including the 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesion. To assess oxidative DNA damage as a mechanism of cisplatin and oxaliplatin cytotoxicity, 8-oxodG-directed base excision repair was stably enhanced in human embryonic kidney cells by FLAG-tagged expression of human oxoguanine glycosylase 1 (alpha-OGG1) or its functional homologue, Escherichia coli formamidopyrimidine glycosylase (fpg). Both drugs increased reactive oxygen species and 8-oxodG levels, and cytotoxicity was decreased by antioxidant pretreatment. Ectopic expression of alpha-OGG1 or fpg in cell clones increased nuclear and mitochondrial 8-oxodG repair, and reduced death by reactive oxygen species initiators (H(2)O(2), menadione) and both platinum drugs. Exposure to oxaliplatin caused a more marked and sustained block of cell proliferation than exposure to cisplatin. We conclude that the 8-oxodG lesion is cytotoxic, and base excision repair a likely determinant of risk. The greater antitumor efficacy of oxaliplatin seems unrelated to oxidative DNA damage, suggesting a novel strategy for improving the therapeutic index in cancer therapy.

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Mutagenic potential of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass catalyzed by human Y-family DNA polymerases.

Project description:One of the most common lesions induced by oxidative DNA damage is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). Replicative DNA polymerases poorly traverse this highly mutagenic lesion, suggesting that the replication fork may switch to a polymerase specialized for translesion DNA synthesis (TLS) to catalyze 8-oxodG bypass in vivo. Here, we systematically compared the 8-oxodG bypass efficiencies and fidelities of the TLS-specialized, human Y-family DNA polymerases eta (hPol?), iota (hPol?), kappa (hPol?), and Rev1 (hRev1) either alone or in combination. Primer extension assays revealed that the times required for hPol?, hRev1, hPol?, and hPol? to bypass 50% of the 8-oxodG lesions encountered (t50(bypass)) were 0.58, 0.86, 108, and 670 s, respectively. Although hRev1 bypassed 8-oxodG efficiently, hRev1 failed to catalyze the extension step of TLS, and a second polymerase was required to extend the lesion bypass products. A high-throughput short oligonucleotide sequencing assay (HT-SOSA) was used to quantify the types and frequencies of incorporation errors produced by the human Y-family DNA polymerases at and near the 8-oxodG site. Although hPol? bypassed 8-oxodG most efficiently, hPol? correctly incorporated dCTP opposite 8-oxodG within only 54.5% of the sequences analyzed. In contrast, hPol? bypassed the lesion least efficiently but correctly incorporated dCTP at a frequency of 65.8% opposite the lesion. The combination of hRev1 and hPol? was most accurate opposite 8-oxodG (92.3%), whereas hPol? alone was the least accurate (18.5%). The t50(bypass) value and correct dCTP incorporation frequency in the presence of an equal molar concentration of all four Y-family enzymes were 0.60 s and 43.5%, respectively. These values are most similar to those of hPol? alone, suggesting that hPol? outcompetes the other three Y-family polymerases to catalyze 8-oxodG bypass in vitro and possibly in vivo.

| S-EPMC4033635 | biostudies-literature

Mutagenesis mechanism of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine.

Project description:Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson-Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polβ decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.

| S-EPMC7229865 | biostudies-literature

Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Project description:Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²? as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (?200-1200 nM). DNA binding is enhanced 34-fold by Mn²? (K(d,DNA) = 27 nM). The pol activity of PrimPol is increased 400-1000-fold by Mn²? compared to Mg²? based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ?100-2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ?1.7-fold more accurate when Mg²? is the cofactor compared to Mn²?. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn²?) to 6-fold (Mg²?) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ?25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ?2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ?0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²?, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.

| S-EPMC4204878 | biostudies-other

Short-term biological variation and Reference change values of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine.

Project description:AimThe DNA and RNA oxidative damage products urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-odGsn) and 8-oxo-7,8-dihydroguanosine (8-oGsn) have potential use in clinical practice. However, biological variation (BV) and reference change values (RCVs) have not been established. The aim of this study was to establish the short-term between-subject BV(CVG ), within-subject BV(CVI ), and RCVs for urinary 8-odGsn and 8-oGsn.MethodsFirst-morning midstream urine specimens were collected from 20 apparently healthy subjects(ten males and ten females) on five consecutive days. 8-odGsn and 8-oGsn were measured using LC-MS/MS, while urine creatinine (U-Cr) was also measured to correct their results. A two-level nested ANOVA was used to estimate the CVI and CVG. RESULTS: The values of CVG for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 31.2%, 39.6%, 35.3%, and 28.8%, respectively, while CVI for them were 40.5%, 9.0%, 33.5%, and 12.1%, respectively. The RCVs for 8-odGsn, 8-odGsn/U-Cr, 8-oGsn, and 8-oGsn/U-Cr were 112.5%, 26.7%, 93.7%, and 36.5%, respectively.ConclusionBV and RCVs were firstly established for 8-oxo-dGsn and 8-oGsn, and can be used in clinical practice.

| S-EPMC7755802 | biostudies-literature

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