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Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens. Clostridioides difficile is an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination of in silico predictions and in vitro experiments to demonstrate that C. difficile employs a bipartite origin of replication that shows DnaA-dependent melting at oriC2, located in the dnaA-dnaN intergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region of C. difficile and contributes to our understanding of the initiation of DNA replication in clostridia.

Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens. Clostridioides difficile is an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination of in silico predictions and in vitro experiments to demonstrate that C. difficile employs a bipartite origin of replication that shows DnaA-dependent melting at oriC2 , located in the dnaA - dnaN intergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region of C. difficile and contributes to our understanding of the initiation of DNA replication in clostridia. [ABSTRACT FROM AUTHOR]

Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [ 32 P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [ 32 P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments., Competing Interests: The authors declare no conflicts of interest or competing interests.