Measurement of oxidized and methylated DNA bases by HPLC with electrochemical detection.
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ABSTRACT:
SUBMITTER: Kaur H
PROVIDER: S-EPMC1217582 | biostudies-other | 1996 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
| S-EPMC5570014 | biostudies-literature
Project description:This review article presents, an overview of the DNA glycosylases that recognize oxidized DNA bases using the Fpg/Nei family of DNA glycosylases as models for how structure can inform function. For example, even though human NEIL1 and the plant and fungal orthologs lack the zinc finger shown to be required for binding, DNA crystal structures revealed a "zincless finger" with the same properties. Moreover, the "lesion recognition loop" is not involved in lesion recognition, rather, it stabilizes 8-oxoG in the active site pocket. Unlike the other Fpg/Nei family members, Neil3 lacks two of the three void-filling residues that stabilize the DNA duplex and interact with the opposite strand to the damage which may account for its preference for lesions in single-stranded DNA. Also single-molecule approaches show that DNA glycosylases search for their substrates in a sea of undamaged DNA by using a wedge residue that is inserted into the DNA helix to probe for the presence of damage.
| S-EPMC3997179 | biostudies-literature
Project description:Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC-MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of ethanethiol produced 8-hydroxyguanine levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the derivatization reaction and adding ethanethiol.
| S-EPMC1219362 | biostudies-other
Project description:DNA glycosylase enzymes recognize and remove structurally distinct modified forms of DNA bases, thereby repairing genomic DNA from chemically induced damage or erasing epigenetic marks. However, these enzymes are often promiscuous, and advanced tools are needed to evaluate and engineer their substrate specificity. Thus, in the present study, we developed a new strategy to rapidly profile the substrate specificity of 8-oxoguanine glycosylases, which cleave biologically relevant oxidized forms of guanine. We monitored the enzymatic excision of fluorophore-labeled oligonucleotides containing synthetic modifications 8-oxoG and FapyG, or G. Using this molecular beacon approach, we identified several hOGG1 mutants with higher specificity for FapyG than 8-oxoG. This approach and the newly synthesized probes will be useful for the characterization of glycosylase substrate specificity and damage excision mechanisms, as well as for evaluating engineered enzymes with altered reactivities.
| S-EPMC9007065 | biostudies-literature
Project description:A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.
| S-EPMC2886154 | biostudies-literature
Project description:The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.
| S-EPMC4646274 | biostudies-literature
Project description:5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence of "reader" proteins that recognize 5hmC would be a logical assumption, and various searches have been undertaken to identify proteins that specifically bind to 5hmC and the other oxidized 5mC bases. However, the existence of definitive 5hmC "readers" has remained unclear and proteins interacting specifically with 5fC or 5caC are also very few. On the other hand, 5hmC is incapable of interacting with a number of proteins that recognize 5mC at CpG sequences, suggesting that 5hmC is an anti-reader modification that may serve to displace 5mC readers from DNA. In this review article, we discuss candidate proteins that may interact with oxidized 5mC bases.
| S-EPMC7007358 | biostudies-literature
Project description:Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.
| S-EPMC3264494 | biostudies-literature
Project description:As a result of their inherent planarity, DNA base radicals generated by one-electron oxidation/reduction or bond cleavage form ?- or ?-radicals. While most DNA base systems form ?-radicals, there are a number of nucleobase analogues such as one-electron-oxidized 6-azauraci1, 6-azacytosine, and 2-thiothymine or one-electron reduced 5-bromouracil that form more reactive ?-radicals. Elucidating the availability of these states within DNA, base radical electronic structure is important to the understanding of the reactivity of DNA base radicals in different environments. In this work, we address this question by the calculation of the relative energies of ?- and ?-radical states in DNA/RNA bases and their analogues. We used density functional theory B3LYP/6-31++G** method to optimize the geometries of ?- and ?-radicals in Cs symmetry (i.e., planar) in the gas phase and in solution using the polarized continuum model (PCM). The calculations predict that ?- and ?-radical states in one-electron-oxidized bases of thymine, T(N3-H)(•), and uracil, U(N3-H)(•), are very close in energy; i.e., the ?-radical is only ca. 4 kcal/mol more stable than the ?-radical. For the one-electron-oxidized radicals of cytosine, C(•+), C(N4-H)(•), adenine, A(•+), A(N6-H)(•), and guanine, G(•+), G(N2-H)(•), G(N1-H)(•), the ?-radicals are ca. 16-41 kcal/mol more stable than their corresponding ?-radicals. Inclusion of solvent (PCM) is found to stabilize the ?- over ?-radical of each of the systems. U(N3-H)(•) with three discrete water molecules in the gas phase is found to form a three-electron ? bond between the N3 atom of uracil and the O atom of a water molecule, but on inclusion of full solvation and discrete hydration, the ?-radical remains most stable.
| S-EPMC3946539 | biostudies-literature
Project description:Circulating cell-free DNA (cfDNA) has been intensively investigated as a diagnostic and prognostic marker for various cancers. In recent years, presence of unmethylated insulin cfDNA in the circulation has been correlated with pancreatic ?-cell death and risk of developing type 1 diabetes. Digital (d)PCR is an increasingly popular method of quantifying insulin cfDNA due to its ability to determine absolute copy numbers, and its increased sensitivity when compared to the more routinely used quantitative PCR. Multiple platforms have been developed to carry out dPCR. However, not all technologies perform comparably, thereby necessitating evaluation of each platform. Here, we compare two dPCR platforms: the QuantStudio 3D (QS3D, Applied Biosystems) and the QX200 (Bio-Rad), to measure copies of unmethylated/methylated insulin plasmids. The QS3D detected greater copy numbers of the plasmids than the QX200 (manual mode), whereas QX200 demonstrated minimal replicate variability, increased throughput, ease of use and the potential for automation. Overall, the performance of QX200, in our hands, was better suited to measure differentially methylated insulin cfDNA.
| S-EPMC6641562 | biostudies-literature