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Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

ABSTRACT: Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites. This method allows direct kinetic measurements by LC-MS/MS sequencing for oligonucleotides longer than 20 base pairs (bp). We used MeC and non-MeC (C) versions of a 32?bp exon 7 fragment of the p53 gene. Methylation of 19 cytosines increased the rate constant 3-fold for adduction on G at the major reactive CpG in codon 248 vs. the non-MeC fragment. Rate constants for non-CpG codons 244 and 243 were not influenced significantly by MeC. Conformational and hydrophobicity changes in the MeC-p53 exon 7 fragment revealed by CD spectra and molecular modeling increase the BPDE binding constant to G in codon 248 consistent with a pathway in which preceding reactant binding greatly facilitates the rate of covalent S_N2 coupling.

SUBMITTER: Malla S

PROVIDER: S-EPMC5244379 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

Malla Spundana S Kadimisetty Karteek K Fu You-Jun YJ Choudhary Dharamainder D Schenkman John B JB Rusling James F JF

Scientific reports 20170119

Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology ...[more]

PMID: 28102315

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Project description:Biomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite.The CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis.In this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element.This study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species.

Dataset Information

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

Publications

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

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