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.5-chloro-deoxycytidine | hypochlorite | myeloperoxidase | inflammatory bowel disease | carcinogenesis

Mutagenic consequences of cytosine alterations site-specifically embedded in the human genome

“…Regarding halogenated cytosine, it has been suggested that 5-ClC causes C:G to T:A transition mutations at rates ranging from 5 to 9 % by mispairing with adenine in E. coli [32]. Based on our results, however, 5-BrC did not induce C:G to T:A transition mutations (0 %, Fig.…”

Occurrence, Biological Consequences, and Human Health Relevance of Oxidative Stress-Induced DNA Damage

“…It was recently reported that all families of DNA polymerases predominantly decode 5-Cl-C as C in vitro ; meanwhile, 5-Cl-C, when placed on single-stranded M13 plasmid and replicated in E. coli cells, induced 3–9% C → T transition, which is as mutagenic as 8-oxo-dG in similar assays. 264 On the other hand, the 5-halogenated derivatives of cytosine may also perturb epigenetic signaling. In this vein, 5–Cl-C and 5–Br-C in a CpG sequence context can, similar to 5-mC, direct DNMT1-mediated maintenance DNA cytosine methylation and can bind to methyl-CpG-binding proteins.…”

Formation and repair of oxidatively generated damage in cellular DNA

“…5-ClCyt is formed predominantly over 5-ClGua and 5-ClAde in the DNA and RNA in SKM-1 cells, with the latter biopolymer showing a higher susceptibility to HOCl reactions [265]. In a subsequent study, the halogenated pyrimidine base 5-ClCyt was higher in the DNA of diabetic patients than in healthy volunteers, suggesting that this product may be used as a relevant biomarker of inflammation [266]. This idea is supported by the [265] observation of a significant increase in the steady-state level of highly mutagenic 5-ClCyt [267] in the colon and liver of Rag2−/− mice that were subjected to chronic inflammation as the result of infection with Helicobacter hepaticus [256].…”