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Oxidized Derivatives of 5-Methylcytosine Alter the Stability and Dehybridization Dynamics of Duplex DNA.

ABSTRACT: The naturally occurring nucleobase 5-methylcytosine (mC) and its oxidized derivatives 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) play important roles in epigenetic regulation and, along with cytosine (C), represent nucleobases currently implicated in the active cytosine demethylation pathway. Despite considerable interest in these modified bases, their impact on the thermodynamic stability of double-stranded DNA (dsDNA) remains ambiguous and their influence on hybridization kinetics and dynamics is even less well-understood. To address these unknowns, we employ steady-state and time-resolved infrared spectroscopy to measure the influence of cytosine modification on the thermodynamics and kinetics of hybridization by assessing the impact on local base pairing dynamics, shifts in the stability of the duplex state, and changes to the hybridization transition state. Modification with mC leads to more tightly bound base pairing below the melting transition and stabilizes the duplex relative to canonical DNA, but the free energy barrier to dehybridization at physiological temperature is nevertheless reduced slightly. Both hmC and fC lead to an increase in local base pair fluctuations, a reduction in the cooperativity of duplex melting, and a lowering of the dissociation barrier, but these effects are most pronounced when the 5-position is formylated. The caC nucleobase demonstrates little impact on dsDNA under neutral conditions, but we find that this modification can dynamically switch between C-like and fC-like behavior depending on the protonation state of the 5-position carboxyl group. Our results provide a consistent thermodynamic and kinetic framework with which to describe the modulation of the physical properties of double-stranded DNA containing these modified nucleobases.

SUBMITTER: Sanstead PJ

PROVIDER: S-EPMC7136776 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Oxidized Derivatives of 5-Methylcytosine Alter the Stability and Dehybridization Dynamics of Duplex DNA.

Sanstead Paul J PJ Ashwood Brennan B Dai Qing Q He Chuan C Tokmakoff Andrei A

The journal of physical chemistry. B 20200205 7

The naturally occurring nucleobase 5-methylcytosine (mC) and its oxidized derivatives 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) play important roles in epigenetic regulation and, along with cytosine (C), represent nucleobases currently implicated in the active cytosine demethylation pathway. Despite considerable interest in these modified bases, their impact on the thermodynamic stability of double-stranded DNA (dsDNA) remains ambiguous and their influenc ...[more]

PMID: 31986043

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Project description:Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC) and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and adult mouse brain tissue. Readers for these modifications are only partially overlapping and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation. Peptides were separated on an EASY-nLC (Proxeon) connected online to an LTQ-Orbitrap-Velos mass spectrometer. Spectra were recorded in CID mode. A gradient of organic solvent (5-30% acetonitrile) was applied (120 minutes) and the top 15 most abundant peptides were fragmented for MS/MS, using an exclusion list of 500 proteins for 45 seconds. Raw data were analyzed using Maxquant version 1.2.2.5 and the integrated Andromeda search engine against protein database ipi.MOUSE.v3.68. Using Perseus, data was filtered for contaminants, reverse hits, number of peptides (>1) and unique peptides (>0). Ratios were logarithmitized (log2) and groups (consisting of forward and reverse) were defined. Proteins were filtered to have at least 2 valid values in one of the groups and missing values were imputed based on a normal distribution (width=0.2 and shift=0), after which Significance B was calculated (Benj.Hoch.FDR=0.05). Scatterplots were made using R. Proteins were defined to be significant when both forward and reverse significance p<0.05 and minimal ratios were >2 in both experiments. The H/L ratios shown in Figure2A-C were calculated using the formula (log(forward ratio) - log(reverse ratio))/2.

Dataset Information

Oxidized Derivatives of 5-Methylcytosine Alter the Stability and Dehybridization Dynamics of Duplex DNA.

Publications

Oxidized Derivatives of 5-Methylcytosine Alter the Stability and Dehybridization Dynamics of Duplex DNA.

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