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Location analysis of 8-oxo-7,8-dihydroguanine in DNA by polymerase-mediated differential coding.

ABSTRACT: Accumulating lines of evidence indicate that reactive oxygen species (ROS) are important signalling molecules for various cellular processes. 8-Oxo-7,8-dihydroguanine (OG) is a prominent oxidative modification formed in DNA by ROS. Recently, it has been proposed that OG may have regulatory and possibly epigenetic-like properties in modulating gene expression by interfering with transcription components or affecting the formation of G-quadruplex structures. Deciphering the molecular mechanisms of OG on regulation of gene expression requires uncovering the location of OG on genome. In the current study, we characterized two commercially available DNA polymerases, Bsu DNA polymerase (Bsu Pol) and Tth DNA polymerase (Tth Pol), which can selectively incorporate adenine (A) and cytosine (C) opposite OG, respectively. By virtue of the differential coding properties of Bsu Pol and Tth Pol that can faithfully or error-prone copy a DNA strand carrying OG, we achieved quantitative and single-base resolution analysis of OG in synthesized DNA that carries OG as well as in the G-rich telomeric DNA from HeLa cells. In addition, the parallel analysis of the primer extension products with Bsu Pol and Tth Pol followed by sequencing provided distinct detection of OG in synthesized DNA. Future application of this approach will greatly increase our knowledge of the chemical biology of OG with respect to its epigenetic-like regulatory roles.

SUBMITTER: Tang F

PROVIDER: S-EPMC6460952 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DNA polymerase ?-interacting protein 2 is a processivity factor for DNA polymerase ? during 8-oxo-7,8-dihydroguanine bypass.

Project description:The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol ?-interacting protein 2 (PolDIP2) has been found to physically interact with Pol ?, Pol ?, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol ? (family B), Pol ? and Pol ? (family Y), and Pol ? and Pol ? (family X). Our results show that PolDIP2 also physically interacts with Pol ?, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols ? and ?, but not by Pols ? or ?. Additionally, we provide evidence that PolDIP2 stimulates Pol ? without affecting its fidelity, facilitating the switch from Pol ? to Pol ? during 8-oxo-G TLS. PolDIP2 stimulates Pols ? and ? mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol ? deficient background, suggesting that PolDIP2 is an important mediator for TLS.

| S-EPMC3839753 | biostudies-literature

DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis.

Project description:The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.

| S-EPMC2593216 | biostudies-other

Trinucleotide repeat DNA alters structure to minimize the thermodynamic impact of 8-oxo-7,8-dihydroguanine.

Project description:In the phenomenon of trinucleotide repeat (TNR) expansion, an important interplay exists between DNA damage repair of 8-oxo-7,8-dihydroguanine (8-oxoG) and noncanonical structure formation. We show that TNR DNA adapts its structure to accommodate 8-oxoG. Using chemical probe analysis, we find that CAG repeats composing the stem-loop arm of a three-way junction alter the population of structures in response to 8-oxoG by positioning the lesion at or near the loop. Furthermore, we find that oligonucleotides composed of odd-numbered repeat sequences, which form populations of two structures, will also alter their structure to place 8-oxoG in the loop. However, sequences with an even number of repeats do not display this behavior. Analysis by differential scanning calorimetry indicates that when the lesion is located within the loop, there are no significant changes to the thermodynamic parameters as compared to the DNA lacking 8-oxoG. This contrasts with the enthalpic destabilization observed when 8-oxoG is base-paired to C and indicates that positioning 8-oxoG in the loop avoids the thermodynamic penalty associated with 8-oxoG base-pairing. Since formation of stem-loop hairpins is proposed to facilitate TNR expansion, these results highlight the importance of defining the structural consequences of DNA damage.

| S-EPMC3254800 | biostudies-literature

Formation of 8-oxo-7,8-dihydroguanine-radicals in gamma-irradiated DNA by multiple one-electron oxidations.

Project description:Electron spin resonance (ESR) studies of radicals formed by radiation-induced multiple one-electron oxidations of guanine moieties in DNA are reported in this work. Annealing of gamma-irradiated DNA from 77 to 235 K results in the hydration of one electron oxidized guanine (G*+) to form the 8-hydroxy-7,8-dihydroguanin-7-yl-radical (*GOH) having one beta-proton coupling of 17-28 G and an anisotropic nitrogen coupling, A(parallel), of approximately 20 G, A(perpendicular) = 0 with g(parallel) = 2.0026 and g(perpendicular) = 2.0037. Further annealing to 258 K results in the formation of a sharp singlet at g = 2.0048 with line-width of 5.3 G that is identified as the 8-oxo-7,8-dihydroguanine one-electron-oxidized radical (8-oxo-G*+). This species is formed via two one-electron oxidations of *GOH. These two one-electron oxidation steps leading to the formation of 8-oxo-G*+ from *GOH in DNA, are in accordance with the expected ease of oxidation of *GOH and 8-oxo-G. The incorporation of oxygen from water in G*+ leading to *GOH and to 8-oxo-G*+ is verified by ESR studies employing 17O isotopically enriched water, which provide unambiguous evidence for the formation of both radicals. ESR analysis of irradiated-DNA in the presence of the electron scavenger, Tl3+, demonstrates that the cationic pathway leads to the formation of the 8-oxo-G*+. In irradiated DNA-Tl3+ samples, Tl3+ captures electrons. Tl2+ thus produced is a strong oxidant (2.2 V), which is metastable at 77 K and is observed to increase the formation of G*+ and subsequently of 8-oxo-G*+ upon annealing. We find that in the absence of the electron scavenger the yield of 8-oxo-G*+ is substantially reduced as a result of electron recombinations with G*+ and possible reaction with *GOH.

| S-EPMC545457 | biostudies-literature

Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site.

Project description:The formation of clustered DNA damage sites is a unique feature of ionizing radiation. Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites. Using a plasmid-based method, damaged DNA containing uracil positioned at 1-5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY. Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions. The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid. In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site. Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC-->TA transversions. The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis.

| S-EPMC373263 | biostudies-literature

Induction of action-at-a-distance mutagenesis by 8-oxo-7,8-dihydroguanine in DNA pol ?-knockdown cells.

Project description:In DNA, 8-oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) is one of the most pivotal oxidatively damaged bases and induces G:C???T:A transversion mutations. DNA polymerase ? preferentially inserts dCTP opposite G(O) in vitro, and this error-free bypass function is considered to be important after A base removal from G(O):A pairs by the MUTYH DNA glycosylase. To examine the effects of reduced levels of DNA polymerase ? on the G(O)-induced mutations, the polymerase was knocked-down in human U2OS cells, and a shuttle plasmid DNA containing a G(O):C pair at position 122 in the supF gene was transfected into the cells. The plasmid DNA replicated in the cells was introduced into an Escherichia coli indicator strain, to measure the supF mutant frequency.The knockdown of DNA polymerase ? significantly enhanced the mutant frequency of the G(O) plasmid DNA. Contrary to our expectations, the knockdown did not promote the targeted G:C???T:A transversion. Instead, substitution mutations at G:C sites other than position 122 were enhanced in the cells.These results suggested that the knockdown of DNA polymerase ? induced action-at-a-distance mutagenesis in human cells when the G(O):C pair was present in the DNA.

| S-EPMC4918004 | biostudies-literature

Human Telomere G-Quadruplexes with Five Repeats Accommodate 8-Oxo-7,8-dihydroguanine by Looping out the DNA Damage.

Project description:Inflammation and oxidative stress generate free radicals that oxidize guanine (G) in DNA to 8-oxo-7,8-dihydroguanine (OG), and this reaction is prominent in the G-rich telomere sequence. In telomeres, OG is not efficiently removed by repair pathways allowing its concentration to build, surprisingly without any immediate negative consequences to stability. Herein, OG was synthesized in five repeats of the human telomere sequence (TTAGGG)n, at the 5'-G of the 5'-most, middle, and 3'-most G tracks, representing hotspots for oxidation. These synthetic oligomers were folded in relevant amounts of K(+)/Na(+) to adopt hybrid G-quadruplex folds. The structural impact of OG was assayed by circular dichroism, thermal melting, (1)H NMR, and single-molecule profiling by the α-hemolysin nanopore. On the basis of these results, OG was well accommodated in the five-repeat sequences by looping out the damaged G track to allow the other four tracks to adopt a hybrid G-quadruplex. These results run counter to previous studies with OG in four-repeat telomere sequences that found OG to be highly destabilizing and causing significant reorientation of the fold. When taking a wider view of the human telomere sequence and considering additional repeats, we found OG to cause minimal impact on the structure. The plasticity of this repeat sequence addresses how OG concentrations can increase in telomeres without immediate telomere instability or attrition.

| S-EPMC4828913 | biostudies-literature

8-Oxo-7,8-dihydroguanine, friend and foe: Epigenetic-like regulator versus initiator of mutagenesis.

Project description:A high flux of reactive oxygen species during oxidative stress results in oxidative modification of cellular components including DNA. Oxidative DNA "damage" to the heterocyclic bases is considered deleterious because polymerases may incorrectly read the modifications causing mutations. A prominent member in this class is the oxidized guanine base 8-oxo-7,8-dihydroguanine (OG) that is moderately mutagenic effecting G→T transversion mutations. Recent reports have identified that formation of OG in G-rich regulatory elements in the promoters of the VEGF, TNFα, and SIRT1 genes can increase transcription via activation of the base excision repair (BER) pathway. Work in our laboratory with the G-rich sequence in the promoter of VEGF concluded that BER drives a shift in structure to a G-quadruplex conformation leading to gene activation in mammalian cells. More specifically, removal of OG from the duplex context by 8-oxoguanine glycosylase 1 (OGG1) produces an abasic site (AP) that destabilizes the duplex, shifting the equilibrium toward the G-quadruplex fold because of preferential extrusion of the AP into a loop. The AP is bound but inefficiently cleaved by apurinic/apyrimidinic endoDNase I (APE1) that likely allows recruitment of activating transcription factors for gene induction. The ability of OG to induce transcription ascribes a regulatory or epigenetic-like role for this oxidatively modified base. We compare OG to the 5-methylcytosine (5mC) epigenetic pathway including its oxidized derivatives, some of which poise genes for transcription while also being substrates for BER. The mutagenic potential of OG to induce only ∼one-third the number of mutations (G→T) compared to deamination of 5mC producing C→T mutations is described. These comparisons blur the line between friendly epigenetic base modifications and those that are foes, i.e. DNA "damage," causing genetic mutations.

| S-EPMC5548303 | biostudies-literature

Sequence-specific single-molecule analysis of 8-oxo-7,8-dihydroguanine lesions in DNA based on unzipping kinetics of complementary probes in ion channel recordings.

Project description:Translocation measurements of intact DNA strands with the ion channel α-hemolysin (α-HL) are limited to single-stranded DNA (ssDNA) experiments as the dimensions of the channel prevent double-stranded DNA (dsDNA) translocation; however, if a short oligodeoxynucleotide is used to interrogate a longer ssDNA strand, it is possible to unzip the duplex region when it is captured in the α-HL vestibule, allowing the longer strand to translocate through the α-HL channel. This unzipping process has a characteristic duration based on the stability of the duplex. Here, ion channel recordings are used to detect the presence and relative location of the oxidized damage site 8-oxo-7,8-dihydroguanine (OG) in a sequence-specific manner. OG engages in base pairing to C or A with unique stabilities relative to native base Watson-Crick pairings, and this phenomenon is used here to engineer probe sequences (10-15mers) that, when base-paired with a 65mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (T(m)). Unzipping times also depend on the direction from which the duplex enters the vestibule if the stabilities of leading base pairs at the ends of the duplex are significantly different. It is shown here that the presence of a single DNA lesion can be distinguished from an undamaged sequence and that the relative location of the damage site can be determined based on the duration of duplex unzipping.

| S-EPMC3173567 | biostudies-literature

Sequencing the Mouse Genome for the Oxidatively Modified Base 8-Oxo-7,8-dihydroguanine by OG-Seq.

Project description:Oxidative damage to the genome can yield the base 8-oxo-7,8-dihydroguanine (OG). In vitro studies suggested OG would preferentially form in 5'-GG-3' sequence contexts after exposure to reactive oxygen species. Herein, OG locations in the genome were studied by development of "OG-Seq" to sequence OG sites via next-generation sequencing at ?0.15-kb resolution. The results of this study found ?10?000 regions of OG enrichment in WT mouse embryonic fibroblasts and ?18?000 regions when the OG repair glycosylase Ogg1 was knocked out. Gene promoters and UTRs harbor more OG-enriched sites than expected if the sites were randomly distributed throughout the genome and correlate with reactive 5'-GG-3' sequences, a result supporting decades of in vitro studies. Sequencing of OG paves the way to address chemical and biological questions surrounding this modified DNA base, such as its role in disease-specific mutations and its epigenetic potential in gene regulation.

| S-EPMC5440228 | biostudies-literature

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