2′-(R)-Fluorinated mC, hmC, fC and caC triphosphates are substrates for DNA polymerases and TET-enzymes

2016 ◽  
Vol 52 (100) ◽  
pp. 14361-14364 ◽  
Author(s):  
A. S. Schröder ◽  
E. Parsa ◽  
K. Iwan ◽  
F. R. Traube ◽  
M. Wallner ◽  
...  
Keyword(s):  
Dna Polymerases ◽  
Dna Bases ◽  
Reaction Pathways ◽  
Tet Enzymes ◽  
In Cells

A deeper investigation of the chemistry that occurs on the newly discovered epigenetic DNA bases 5-hydroxymethyl-(hmdC), 5-formyl-(fdC), and 5-carboxy-deoxycytidine (cadC) requires chemical tool compounds, which are able to dissect the different potential reaction pathways in cells.

scholarly journals Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome

2020 ◽  
Vol 117 (21) ◽  
pp. 11409-11420 ◽  
Author(s):  
Shrabasti Roychoudhury ◽  
Suravi Pramanik ◽  
Hannah L. Harris ◽  
Mason Tarpley ◽  
Aniruddha Sarkar ◽  
...  
Keyword(s):  
Excision Repair ◽  
Factor Loading ◽  
Telomere Maintenance ◽  
Epigenetic Mechanism ◽  
Dna Bases ◽  
G Quadruplex ◽  
Base Excision ◽  
Ap Site ◽  
In Cells

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

scholarly journals Beyond the Lesion: Back to High Fidelity DNA Synthesis

2022 ◽  
Vol 8 ◽  
Author(s):  
Joseph D. Kaszubowski ◽  
Michael A. Trakselis
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Structural Features ◽  
Dna Bases ◽  
High Fidelity ◽  
Nucleotide Incorporation ◽  
Hand Off ◽  
Enzymatic Mechanisms

High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.

Formation of Modified DNA Bases in Cells Exposed either to Gamma Radiation or to High-LET Particles1

2002 ◽  
Vol 157 (5) ◽  
pp. 589-595 ◽  
Author(s):  
J-P. Pouget ◽  
S. Frelon ◽  
J-L. Ravanat ◽  
I. Testard ◽  
F. Odin ◽  
...  
Keyword(s):  
Gamma Radiation ◽  
Dna Bases ◽  
Modified Dna ◽  
High Let ◽  
In Cells

Use of DNA Repair Enzymes in Electrochemical Detection of Damage to DNA Bases in Vitro and in Cells

2005 ◽  
Vol 77 (9) ◽  
pp. 2920-2927 ◽  
Author(s):  
Kateřina Cahová-Kuchaříková ◽  
Miroslav Fojta ◽  
Tomáš Mozga ◽  
Emil Paleček
Keyword(s):  
Dna Repair ◽  
Electrochemical Detection ◽  
Dna Bases ◽  
Dna Repair Enzymes ◽  
Repair Enzymes ◽  
In Cells

scholarly journals MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

2019 ◽  
Author(s):  
Sean G Rudd ◽  
Helge Gad ◽  
Nuno Amaral ◽  
Anna Hagenkort ◽  
Petra Groth ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Mitotic Cell ◽  
Mitotic Arrest ◽  
Oxygen Species ◽  
Mitotic Progression ◽  
Dna Polymerase Delta ◽  
Replication Fork Progression ◽  
In Cells

ABSTRACTReactive oxygen species (ROS) oxidise nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidised nucleotides by preventing incorporation via the oxidised purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidised nucleotides in cells or how oxidised nucleotides in DNA become toxic. We show replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), to an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with the MTH1 inhibitor (MTH1i) TH588. The resulting elevated genomic 8-oxodG correlates with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression is observed, but rather mitotic progression is impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevents accumulation of genomic 8-oxodG and reduces cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS build-up and oxidation of the nucleotide pool. Furthermore, we demonstrate delayed mitosis and increased mitotic cell death following TH588 treatment in cells expressing the error-prone Pol δ variant, which is not observed following treatments with anti-mitotic agents, thus linking incorporation of oxidised nucleotides and disturbed mitotic progression.

scholarly journals A MYC Driven and Metabolite Executed Axis Controls the Subcellular Localization and Activity of TET DNA Hydroxylases and RNA Demethylases in B Cell Lymphomas

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 114-114
Author(s):  
Zhijun Qiu ◽  
An-Ping Lin ◽  
Shoulei Jiang ◽  
Binu Sasi ◽  
Patricia Dahia ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Cellular Localization ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Primary Tumors ◽  
Intermediate Metabolites ◽  
Tet Enzymes ◽  
In Cells

Mitochondria play a role in epigenetic remodeling by generating intermediate metabolites (αKG, 2-HG, succinate, etc.) and modulating the activity of enzymes that control DNA, RNA and histone demethylation. The mitochondrial dehydrogenases D2HGDH and L2HGDH regulate 2-HG/αKG homeostasis by catalyzing the oxidation of D2-HG and L2-HG, respectively, into αKG. In humans, loss of either enzyme causes neuro-metabolic syndromes and early mortality, pointing to their essential role in physiology. Yet, the regulation of D2HGDH and L2HGDH remains to be characterized. To address this issue, we mapped the D2HGDH and L2HGDH promoter regions. Using reporter and ChIP assays, lymphoma cell lines and a mouse model, we discovered that MYC binds to E-boxes in these promoters and directly induce D2HGDH and L2HGDH transcription. We created CRISPR KOs of D2HGDH or L2HGDH in MYC-inducible P493-6 cells, and quantified αKG, D2-HG and L2-HG by LC/MS. We found that turning MYC expression ON led to significantly higher accumulation of αKG (and decrease in 2-HG) in cells expressing D2/L2HGDH than in the KO models, confirming that MYC's transcriptional activation of D2/L2HGDH directly influences 2-HG/αKG balance. Then, we tested whether the metabolic output of the MYC-D2/L2HGDH axis influenced the activity of the αKG-dependent TET enzymes, and of the RNA demethylases FTO and ALKBH5. In P493-6 cells, B cells from Eμ-Myc mice and DLBCL cell lines, MYC expression led to a significant increase in 5hmC, decrease in 5mC DNA, and reduction in m6A RNA marks. These MYC-induced changes were blunted in D2HGDH or L2HGDH KO cells. In addition, the effects of MYC on 5hmC/5mC and m6A were blocked in cells exposed to cell-permeable D2-HG or L2-HG, while synthetic αKG rescued D2HGDH and L2HGDH KO cells. Next, we quantified TET and FTO/ALKBH5 activity in the nuclear lysates of our models. In all instances, MYC significantly elevated TET/FTO/ALKBH5 activity in D2HGDH/L2HGDH WT cells, an outcome that was significantly dampened in the KO models. These data suggested that the MYC-D2/L2HGDH axis increases the activity of these enzymes by generating αKG. To examine whether additional layers of control were also operational, we quantified the expression of TET1-3, FTO and ALKBH5, and of members of the RNA methyltransferase complex, METLL3, METLL14 and WTAP, and confirmed that their global expression were unmodified by MYC or other genetic and metabolic perturbations. Strikingly, however, when examining subcellular localization, we found that MYC, in a D2/L2HGDH dependent manner, promoted the nuclear localization of TET1-3, FTO and ALKBH5 (but not METTL3/14). Remarkably, these effects were readily recapitulated by αKG, whereas 2-HG retained these proteins in the cytoplasm, effectively reducing their ability to chemically modify DNA and RNA. Lastly, to test if the MYC-driven increase of TET activity was also present in primary tumors, we explored the DLBCL TCGA dataset. We mapped all enhancers that are active in DLBCL and found that enhancer hypomethylation (a putative consequence of TET activity) significantly correlated with MYC expression (r=0.38, p=0.009) or activity (ssGSEA-derived MYC activity score, r=0.46, p=0.0013); this finding remained significant after excluding potentially cofounding TET2-mutant DLBCLs. We next tested if this MYC-driven/TET-mediated enhancer demethylation was functionally relevant by correlating it to the expression of the gene closest to, or overlapping with, the enhancer. A biologically coherent negative correlation between enhancer methylation and target transcript expression was found for multiple genes, including several products previously implicated in lymphoma pathogenesis, including, FOXP1, PIM1, ATF5, KLHL14 and BRD2. In summary, we showed that D2HGDH and L2HGDH are transcriptional targets of MYC, and that the control of αKG levels by the MYC-D2/L2HGDH axis activates TETs and RNA demethylases. We discovered that MYC and intermediate metabolites control the sub-cellular localization of these enzymes, possibly via covalent modifications, thus adding an new layer of complexity to the remodeling of the epigenome and epitranscriptome in cancer. We showed in primary DLBCLs that MYC expression/activity correlates with hypomethylated/active oncogenic enhancers. Thus, we postulate that downstream to MYC TET enzymes may in specific contexts function as oncogenes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals

1998 ◽  
Vol 335 (2) ◽  
pp. 233-240 ◽  
Author(s):  
Tiberiu SIMANDAN ◽  
Jing SUN ◽  
Thomas A. DIX
Keyword(s):  
Oxidative Damage ◽  
Cellular Systems ◽  
Peroxyl Radicals ◽  
Dna Bases ◽  
Molecular Probe ◽  
Mutagenic Potential ◽  
Dna Base ◽  
Disease Initiation ◽  
Cellular Dna ◽  
In Cells

DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO•) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO• oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO• radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO•) oxidations in cells. ROO• radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO• radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO• oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO• in cellular systems.

scholarly journals Roles of YqjH and YqjW, Homologs of the Escherichiacoli UmuC/DinB or Y Superfamily of DNA Polymerases, in Stationary-Phase Mutagenesis and UV-Induced Mutagenesis of Bacillussubtilis

2003 ◽  
Vol 185 (7) ◽  
pp. 2153-2160 ◽  
Author(s):  
Huang-Mo Sung ◽  
Gabriel Yeamans ◽  
Christian A. Ross ◽  
Ronald E. Yasbin
Keyword(s):  
Stationary Phase ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Protein Structures ◽  
Protein Analysis ◽  
Dna Polymerase V ◽  
Induced Mutagenesis ◽  
Dna Polymerase Iv ◽  
In Cells

ABSTRACT YqjH and YqjW are Bacillus subtilis homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in Escherichia coli. While the functions of YqjH and YqjW in B. subtilis are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to E. coli DNA polymerase IV (DinB protein), and YqjW has 26% identity to E. coli DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the yqjW operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of B. subtilis to generate revertants at the hisC952 allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.

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