DNA | September 2021 - Browse Articles

11 pages, 2505 KiB

Open AccessArticle

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

by Aninda Sundar Dey, Chayan Bhattacharya, Yihong Guan, Babal Kant Jha and Mridul Mukherji

DNA 2021, 1(1), 26-36; https://0-doi-org.brum.beds.ac.uk/10.3390/dna1010004 - 21 Sep 2021

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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 [...] Read more.

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. Full article

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13 pages, 2324 KiB

Open AccessArticle

Ligase A and RNase HI Participate in Completing Replication on the Chromosome in Escherichia coli

by Brian M. Wendel, Adrian J. Hernandez, Charmain T. Courcelle and Justin Courcelle

DNA 2021, 1(1), 13-25; https://0-doi-org.brum.beds.ac.uk/10.3390/dna1010003 - 03 Sep 2021

Cited by 2 | Viewed by 2863

Abstract

In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. [...] Read more.

In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. Two steps predicted to be necessary to complete replication are removal of Okazaki RNA fragments and ligation of the nascent strands at convergent replication forks. E. coli encodes two RNases that remove RNA-DNA hybrids, rnhA and rnhB, as well as two ligases, ligA and ligB. Here, we used replication profiling to show that rnhA and ligA, encoding RNase HI and Ligase A, participate in the completion reaction. Deletion of rnhA impaired the ability to complete replication and resulted in over-replication in the terminus region. It additionally suppressed initiation events from oriC, suggesting a role for the enzyme in oriC-dependent initiation, as has been suggested previously. We also show that a temperature-sensitive mutation in Ligase A led to over-replication at sites where replication completes, and that degradation at these sites occurred upon shifting to the nonpermissive temperature. Deletion of rnhB or ligB did not affect the growth or profile of replication on the genome. Full article

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10 pages, 813 KiB

Open AccessReview

The Pre-Metaphase Stretch: A Re-Examination

by Megan A. Czekalski and Leocadia V. Paliulis

DNA 2021, 1(1), 3-12; https://0-doi-org.brum.beds.ac.uk/10.3390/dna1010002 - 29 Jun 2021

Cited by 1 | Viewed by 3838

Abstract

Pre-metaphase stretch is a term first coined by the preeminent cell biologist Sally Hughes-Schrader in 1950 to describe an elongation of prometaphase chromosomes observed in the primary spermatocytes of phasmid insects and praying mantids. Research from many groups since Hughes-Schrader’s initial observation has [...] Read more.

Pre-metaphase stretch is a term first coined by the preeminent cell biologist Sally Hughes-Schrader in 1950 to describe an elongation of prometaphase chromosomes observed in the primary spermatocytes of phasmid insects and praying mantids. Research from many groups since Hughes-Schrader’s initial observation has revealed reasons for both how and why chromosomes might elongate prior to metaphase. In this review, we describe Hughes-Schrader’s initial findings and discuss how recent work illuminates and provides some mechanistic explanation for this long-ago observed phenomenon. Full article

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