scholarly journals DNA Demethylation in the Processes of Repair and Epigenetic Regulation Performed by 2-Ketoglutarate-Dependent DNA Dioxygenases

2021 ◽  
Vol 22 (19) ◽  
pp. 10540
Author(s):  
Nikita A. Kuznetsov ◽  
Lyubov Yu. Kanazhevskaya ◽  
Olga S. Fedorova
Keyword(s):  
Dna Adducts ◽  
Epigenetic Regulation ◽  
Catalytic Mechanism ◽  
Regulation Of Gene Expression ◽  
Dna Demethylation ◽  
Epigenetic Mark ◽  
Oxidative Demethylation ◽  
Methylation Of Dna ◽  
Alkyl Groups ◽  
Main Pathway

Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate–dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1–8 and TET1–3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).

scholarly journals TET2 coactivates gene expression through demethylation of enhancers

2018 ◽  
Vol 4 (11) ◽  
pp. eaau6986 ◽  
Author(s):  
Lu Wang ◽  
Patrick A. Ozark ◽  
Edwin R. Smith ◽  
Zibo Zhao ◽  
Stacy A. Marshall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regulation Of Gene Expression ◽  
Estrogen Receptor Α ◽  
Dna Demethylation ◽  
Estrogen Response ◽  
Dna Base ◽  
Modified Dna ◽  
Global Increase ◽  
Methylation Patterns

The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

scholarly journals Thymine DNA Glycosylase Can Rapidly Excise 5-Formylcytosine and 5-Carboxylcytosine

2011 ◽  
Vol 286 (41) ◽  
pp. 35334-35338 ◽  
Author(s):  
Atanu Maiti ◽  
Alexander C. Drohat
Keyword(s):  
Embryonic Development ◽  
Catalytic Mechanism ◽  
Excision Repair ◽  
Dna Demethylation ◽  
Oxidation Products ◽  
Dna Glycosylase ◽  
Thymine Dna Glycosylase ◽  
Active Demethylation ◽  
Active Dna Demethylation ◽  
Base Excision

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.

scholarly journals Degradation of 5hmC-marked stalled replication forks by APE1 causes genomic instability

2020 ◽  
Vol 13 (645) ◽  
pp. eaba8091 ◽  
Author(s):  
Suhas S. Kharat ◽  
Xia Ding ◽  
Divya Swaminathan ◽  
Akshey Suresh ◽  
Manish Singh ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Excision Repair ◽  
Human Cancer ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Parp Inhibition ◽  
Epigenetic Mark ◽  
Replication Forks ◽  
A Genome ◽  
Stalled Replication Forks

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair–associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.

scholarly journals Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

2020 ◽  
Vol 48 (8) ◽  
pp. 4041-4051 ◽  
Author(s):  
Xiwen Xing ◽  
Shinsuke Sato ◽  
Nai-Kei Wong ◽  
Kumi Hidaka ◽  
Hiroshi Sugiyama ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Excision Repair ◽  
Regulation Of Gene Expression ◽  
Dna Demethylation ◽  
Dna Origami ◽  
Enzymatic Reactions ◽  
Oxidation Reactions ◽  
Methyl Cytosine ◽  
Tet Enzymes

Abstract DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.

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