Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID

From Alpha to Delta—Genetic Epidemiology of SARS-CoV-2 (hCoV-19) in Southern Poland

In Poland, the first case of SARS-CoV-2 infection was confirmed in March 2020. Since then, many circulating virus lineages fueled rapid pandemic waves which inflicted a severe burden on the Polish healthcare system. Some of these lineages were associated with increased transmissibility and immune escape. Mutations in the viral spike protein, which is responsible for host cell recognition and serves as the primary target for neutralizing antibodies, are of particular importance. We investigated the molecular epidemiology of the SARS-CoV-2 clades circulating in Southern Poland from February 2021 to August 2021. The 921 whole-genome sequences were used for variant identification, spike mutation, and phylogenetic analyses. The Pango B.1.1.7 was the dominant variant (n = 730, 89.68%) from March 2021 to July 2021. In July 2021, the B.1.1.7 was displaced by the B.1.617.2 lineage with 66.66% in July 2021 and 92.3% in August 2021 frequencies, respectively. Moreover, our results were compared with the sequencing available on the GISAID platform for other regions of Poland, the Czech Republic, and Slovakia. The analysis showed that the dominant variant in the analyzed period was B.1.1.7 in all countries and Southern Poland (Silesia). Interestingly, B.1.1.7 was replaced by B.1.617.2 earlier in Southern Poland than in the rest of the country. Moreover, in the Czech Republic and Slovakia, AY lineages were predominant at that time, contrary to the Silesia region