Active demethylation of 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER). The TDG-BER pathway may lead to the generation of DNA strand breaks, potentially compromising genome integrity. Alternatively, direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks. However, cleavage of the C-C bond in 5caC in human cells remains an open question. We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate. After incubation with whole-cell protein extract or transfection into human cells, we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at both the mononucleotide and oligodeoxynucleotide levels. Our results clearly showed the direct conversion of 5caC to cytosine in human cells, providing evidence to support a novel pathway for active DNA demethylation.

The report of the existence of 5-hydroxymethylcytosine (hm 5 C) in mammalian genomes is a milestone discovery. hm 5 C is now generally viewed as the sixth base of DNA with important functions on epigenetic regulation. The in-depth investigation of the biological functions of hm 5 C requires elucidating the distribution patterns of hm 5 C in genomes, better in single-nucleotide resolution. It was reported that the cytosine deaminases of the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide-like) family are nucleic acid editing enzymes and can deaminate cytosine (C) to form uracil (U). Particularly, a subfamily of APOBEC (APOBEC3A) can efficiently deaminate both C and 5methylcytosine (m 5 C). In the current study, we identified that APOBEC3A protein can effectively deaminate C, m 5 C, and hm 5 C but shows no observable deamination activity toward glycosylated hm 5 C (β-glucosyl-5-hydroxymethyl-2′-deoxycytidine, ghm 5 C) by using the restriction enzyme-based assay and liquid chromatography−electrospray ionization− tandem mass spectrometry (LC-ESI-MS/MS) analysis. By virtue of the differential deamination activity of APOBEC3A toward C, m 5 C, and ghm 5 C in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hm 5 C in DNA. In this analytical strategy, the original C and m 5 C in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghm 5 C is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hm 5 C, which offers the single-nucleotide resolution analysis of hm 5 C in DNA. This APOBEC3A-mediated deamination sequencing (AMD-seq) is straightforward and involves no bisulfite treatment, which avoids the substantial degradation of DNA. Future application of this strategy can be performed for the reliable mapping of hm 5 C in genome-wide scale at the single-nucleotide resolution.

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by the writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible...

The discovery of 5-hydroxymethylcytosine (5hmC) in mammalian genomes is a landmark in epigenomics study. Similar to 5-methylcytosine (5mC), 5hmC is viewed a critical epigenetic modification. Deciphering the functions of 5hmC...

Natural plasmid transformation of Escherichia coli is a complex process that occurs strictly on agar plates and requires the global stress response factor S . Here, we showed that additional carbon sources could significantly enhance the transformability of E. coli. Inactivation of phosphotransferase system genes (ptsH, ptsG, and crr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed that crp and cyaA mutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed that rpoS is negatively regulated by cAMP-CRP in early log phase and that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. Therefore, it can be concluded that both the crp and cyaA mutations derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressing rpoS expression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation in E. coli. IMPORTANCEEscherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and antibiotic resistance genes. In this work, we found that carbon sources significantly improve transformation by decreasing cAMP. Then, the low level of cAMP-CRP derepresses the general stress response regulator RpoS via a biphasic regulatory pattern, thereby contributing to transformation. Thus, we demonstrate the mechanism by which carbon sources affect natural transformation, which is important for revealing information about the interplay between nutrition state and competence development in E. coli. Horizontal gene transfer (HGT) is the transfer of genes between distantly related organisms. It is widely recognized that HGT contributes significantly to the evolution of bacterial genomes and the adaptation of bacteria to new environments (1, 2). In bacteria, the physical process of DNA transfer is accomplished by transduction, conjugation, and transformation. Natural transformation is characterized by the spontaneous uptake of free DNA from the environment by a competent cell, which then integrates said DNA into its chromosome or stabilizes the DNA extr...