title: 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 SN2 coupling.
authors.affiliation: University of Connecticut, Department of Chemistry, Storrs, CT 06269, USA.
authors.forename: Spundana
authors.initials: S
authors.lastname: Malla
authors.affiliation: University of Connecticut, Department of Chemistry, Storrs, CT 06269, USA.
authors.forename: Karteek
authors.initials: K
authors.lastname: Kadimisetty
authors.affiliation: University of Connecticut, Department of Chemistry, Storrs, CT 06269, USA.
authors.forename: You-Jun
authors.initials: YJ
authors.lastname: Fu
authors.affiliation: UConn, UConn Health, Department of Surgery and Neag Cancer Center, Farmington, CT 06032, USA.
authors.forename: Dharamainder
authors.initials: D
authors.lastname: Choudhary
authors.affiliation: Professor Emeritus, UConn Health, Farmington, CT 06269, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Schenkman
authors.affiliation: University of Connecticut, Department of Chemistry, Storrs, CT 06269, USA.|UConn, UConn Health, Department of Surgery and Neag Cancer Center, Farmington, CT 06032, USA.|University of Connecticut, Institute of Material Science, Storrs, CT 06269, USA.|School of Chemistry, National University of Ireland at Galway, School of Chemistry, Ireland.
authors.forename: James F
authors.initials: JF
authors.lastname: Rusling
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018899:CpG Islands; D003596:Cytosine / Q000031:analogs & derivatives / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis*; D005091:Exons; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D009690:Nucleic Acid Conformation; D053719:Tandem Mass Spectrometry; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
chemical_list: D018736:DNA Adducts; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D003596:Cytosine