SRP469967 PRJNA1035149 Mutational spectrum produced by 5-chloro-2'-deoxycytidine The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissue. Replication of a site-specific 5ClC in a viral DNA genome results in C>T mutations, consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, non-mutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool, by exposing cells to the nucleoside 5-chloro-2'-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. Duplex sequencing of DNA from MEFs derived from the gpt-delta C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG>TA transitions in 5'-GC(Y)-3' contexts (Y = a pyrimidine) were dominant, while TA>CG transitions appeared at a lower frequency. The mutational spectrum uncovered presented notable similarity to COSMIC mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively.