title: Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer.
abstract: During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase β replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Fedeles
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
authors.forename: Bret D
authors.initials: BD
authors.lastname: Freudenthal
authors.affiliation: Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Emily
authors.initials: E
authors.lastname: Yau
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Vipender
authors.initials: V
authors.lastname: Singh
authors.affiliation: Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Shiou-chi
authors.initials: SC
authors.lastname: Chang
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Deyu
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: James C
authors.initials: JC
authors.lastname: Delaney
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; Department Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; jessig@mit.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Essigmann
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism; D063646:Carcinogenesis; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004252:DNA Mutational Analysis; D006801:Humans; D006997:Hypochlorous Acid / Q000737:chemistry; D007249:Inflammation / Q000378:metabolism; D015212:Inflammatory Bowel Diseases / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009153:Mutagens; D009154:Mutation; D009369:Neoplasms / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D009195:Peroxidase / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D014408:Biomarkers, Tumor; D009153:Mutagens; D009841:Oligonucleotides; D006997:Hypochlorous Acid; D003596:Cytosine; D009195:Peroxidase; C401182:5-chlorocytosine
keywords: 5-chloro-deoxycytidine; carcinogenesis; hypochlorite; inflammatory bowel disease; myeloperoxidase