Abstract
5-methylcytosine (5-mC) can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and finally to 5-carboxylcytosine (5-caC), which is thought to function in active DNA cytosine demethylation in mammals. Although the roles of 5-mC in epigenetic regulation of gene expression are well established, the effects of 5-hmC, 5-foC and 5-caC on DNA replication remain unclear. Here we report a systematic study on how these cytosine derivatives (5-hmC, 5-foC and 5-caC) perturb the efficiency and accuracy of DNA replication using shuttle vector technology in conjugation with next-g sequencing. Our results demonstrated that, in Escherichia coli cells, all the cytosine derivatives could induce CT transition mutation at frequencies of 0.17%–1.12%, though no effect on replication efficiency was observed. These findings provide an important new insight on the potential mutagenic properties of cytosine derivatives occurring as the intermediates of DNA demethylation.
Highlights
Every single cell in a living organism carries the genome, which functions for the storage, replication and transmission of the genetic information
To illustrate the roles of various translesion synthesis DNA polymerases in bypassing these cytosine derivatives in vivo, we employed wild-type AB1157 E. coli cells as well as the isogenic strains deficient in pol II, pol IV, pol V, or both pol IV and pol V as the host cells for the replication study
The sequencing reads obtained for the cytosine derivative-containing genomes relative to control genomes allowed for the calculation of bypass efficiencies for the cytosine derivative
Summary
Every single cell in a living organism carries the genome, which functions for the storage, replication and transmission of the genetic information. Cytosine methylation (5-methylcytosine, 5-mC) at CpG dinucleotide site is the best-characterized epigenetic mark involved in regulating many cellular processes, including embryogenesis, regulation of gene expression, genomic imprinting and X-chromosome inactivation [2]. Consistent with these important roles, a variety of human diseases have been found to be associated with aberrant DNA methylation [3,4]. Active DNA demethylation may be achieved through a multi-step oxidation of 5-mC with the generation of three intermediates, 5hmC, 5-foC and 5-caC (Figure 1)
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