scholarly journals Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase

DNA ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 26-36
Author(s):  
Aninda Sundar Dey ◽  
Chayan Bhattacharya ◽  
Yihong Guan ◽  
Babal Kant Jha ◽  
Mridul Mukherji
Keyword(s):  
Cytosine Methylation ◽  
Mammalian Genome ◽  
Myeloid Malignancies ◽  
Active Demethylation ◽  
Cpg Sites ◽  
Double Stranded Dna ◽  
Demethylation Pathway

In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. Little is known about the enzyme responsible for active demethylation of 5mCpH sites. Using a very sensitive and quantitative LC–MS/MS method, we demonstrate that the human TET2, an iron (II)- and 2OG-dependent dioxygenase, which is a frequently mutated gene in several myeloid malignancies, as well as in a number of other types of cancers, can oxidize 5mCpH sites in double-stranded DNA in vitro. Similar to oxidation of 5mCpG, oxidation of 5mC at CpH sites produces 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) bases in DNA. After 5mCpG, which is the most preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and then 5mCpT. Since the TDG/BER pathway and deformylation or decarboxylation of 5fC or 5caC, respectively, can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase.

scholarly journals Positive/negative ion-switching-based LC–MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Aninda Sundar Dey ◽  
Navid J Ayon ◽  
Chayan Bhattacharya ◽  
William G Gutheil ◽  
Mridul Mukherji
Keyword(s):  
Mass Spectrometry ◽  
Cytosine Methylation ◽  
Negative Ion ◽  
Tandem Mass ◽  
Active Demethylation ◽  
Chromatography Tandem Mass Spectrometry ◽  
In Vitro Characterization ◽  
Liquid Chromatography Tandem Mass

Abstract Cytosine methylation at carbon-5 (5mC) in DNA plays crucial roles in epigenetic transcriptional regulation during metazoan development. The iron (II), 2-oxoglutarate-dependent Ten-Eleven Translocation (TET)-family dioxygenases initiate active demethylation of 5mC. TET2 oxidizes 5mC in nucleic acids into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite the established and emerging roles of TET oxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still in rudimentary stages. Here, we describe an improved positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC–MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. This method will help in further elucidate the function of epigenetically important cytosine modifications. To the best of our knowledge, this is the first study reporting ion-switching-based LC–MS/MS method to analyse cytosine variants produced in TET catalysed reactions.

scholarly journals Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences.

1989 ◽  
Vol 9 (7) ◽  
pp. 2818-2827 ◽  
Author(s):  
C Goyon ◽  
G Faugeron
Keyword(s):  
Dna Sequences ◽  
Cytosine Methylation ◽  
De Novo ◽  
Wild Type Allele ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Type Allele ◽  
Double Stranded Dna ◽  
Ascobolus Immersus

To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.

Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences

1989 ◽  
Vol 9 (7) ◽  
pp. 2818-2827
Author(s):  
C Goyon ◽  
G Faugeron
Keyword(s):  
Dna Sequences ◽  
Cytosine Methylation ◽  
De Novo ◽  
Wild Type Allele ◽  
Wild Type ◽  
Single Stranded Dna ◽  
Type Allele ◽  
Double Stranded Dna ◽  
Ascobolus Immersus

To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.

Discrimination of monomer from multimer DNA disk-shaped toruses by freezeetch TEM

1984 ◽  
Vol 42 ◽  
pp. 210-211
Author(s):  
George C. Ruben ◽  
Kenneth A. Marx
Keyword(s):  
Biochemical Data ◽  
Dna Structures ◽  
Ice Surface ◽  
Double Stranded Dna ◽  
Data Support ◽  
Dna Organization ◽  
Trivalent Cations

In vitro collapse of DNA by trivalent cations like spermidine produces torus (donut) shaped DNA structures thought to have a DNA organization similar to certain double stranded DNA bacteriophage and viruses. This has prompted our studies of these structures using freeze-etch low Pt-C metal (9Å) replica TEM. With a variety of DNAs the TEM and biochemical data support a circumferential DNA winding model for hydrated DNA torus organization. Since toruses are almost invariably oriented nearly horizontal to the ice surface one of the most accessible parameters of a torus population is annulus (ring) thickness. We have tabulated this parameter for populations of both nicked, circular (Fig. 1: n=63) and linear (n=40: data not shown) ϕX-174 DNA toruses. In both cases, as can be noted in Fig. 1, there appears to be a compact grouping of toruses possessing smaller dimensions separated from a dispersed population possessing considerably larger dimensions.

Torus Circumference and Thickness Parameters From a Linear DNA Size Series Suggest How Toruses Self-Assemble

1985 ◽  
Vol 43 ◽  
pp. 522-523
Author(s):  
George C. Ruben ◽  
Kenneth A. Marx
Keyword(s):  
Tertiary Structure ◽  
Dna Complexes ◽  
Tertiary Structures ◽  
Self Assembling ◽  
Double Stranded Dna ◽  
Calf Thymus ◽  
Dna Organization ◽  
Condensed Dna ◽  
Dna Size

Certain double stranded DNA bacteriophage and viruses are thought to have their DNA organized into large torus shaped structures. Morphologically, these poorly understood biological DNA tertiary structures resemble spermidine-condensed DNA complexes formed in vitro in the total absence of other macromolecules normally synthesized by the pathogens for the purpose of their own DNA packaging. Therefore, we have studied the tertiary structure of these self-assembling torus shaped spermidine- DNA complexes in a series of reports. Using freeze-etch, low Pt-C metal (10-15Å) replicas, we have visualized the microscopic DNA organization of both calf Thymus( CT) and linear 0X-174 RFII DNA toruses. In these structures DNA is circumferentially wound, continuously, around the torus into a semi-crystalline, hexagonal packed array of parallel DNA helix sections.

scholarly journals Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

1992 ◽  
Vol 267 (1) ◽  
pp. 331-338
Author(s):  
A J Griffith ◽  
P R Blier ◽  
T Mimori ◽  
J A Hardin
Keyword(s):  
Double Stranded Dna

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

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