Journal: Nature Communications

Article Title: Rad52 mediates class-switch DNA recombination to IgD

doi: 10.1038/s41467-022-28576-2

Figure Lengend Snippet: a Amplified DNAs from junctional intra-σδ deletions as well as Sμ–σδ, Sμ–Sγ1 and Sμ–Sα1 recombinations from human tonsil B cells or human peripheral blood naïve IgM + IgD + B cells stimulated with CpG plus IL-2 and IL-21 and cultured for 120 h, OVA-immunized C57BL/6 mouse spleen B cells or C57BL/6 mouse naïve IgM + IgD + B cells stimulated with LPS plus IL-4 and cultured for 96 h were amplified and sequenced by MiSeq. The length and numbers of nucleotide overlaps (microhomologies) in intra-σδ deletions, Sμ–σδ, Sμ–Sγ1, and Sμ–Sα1 junctional DNAs are shown by violin plots. Each dot represents a unique junctional sequence ( n = 45 per group). b Human and mouse Sμ and σδ regions consist of repetitive motifs, which are better-suited substrates for Rad52-mediated MMEJ than those in Sμ and Sγ1 or Sμ and Sα. As such, they can facilitate the formation of microhomologies. Repetitive sequence elements in mouse and human Sμ, σδ, Sγ1 and Sα that can potentially form microhomologies were identified by Pustell Matrix dot plot using MacVector software and are depicted by small dots. Intensity of dots depicts frequency and degree of complementarity of respective sequences. c Somatic point-mutations in Sμ and σδ regions abetting recombined Sμ−σδ DNA junctions in IgD class-switched human and mouse B cells in vivo and in vitro. Mutations were identified in a 48–506 nt stretch of Sμ or σδ regions in unique Sμ–σδ DNA recombination sequences. Each dot represents an individual sequence. Sequence data were pooled from three individuals in each group. Box and whiskers plots show median, quartiles, maximum and minimum of mutation frequencies in Sμ and σδ regions. In pie charts, the size of slices denotes the proportion of transcripts with the same number of mutations and the gray hue denotes the number of point mutations per transcript. Center of pie shows the total number of independent sequences analyzed. Below the pie charts is the overall mutation frequency (change/base). ** p < 0.01, *** p < 0.001, ns: not significant (unpaired two-tailed t- test). Source data are provided as a Source Data file.

Article Snippet: Consistent with the greatest occurrence of microhomologies in Sμ–σδ junctions, Sμ is better suited for complementary DNA single-strand annealing with σδ than Sγ1 or, to a lesser extent, Sα (mouse) or Sα1 (human), based on various numbers and contexts of these DNA regions discrete motifs, such as [G n ]AGCT repeats (Sμ, Sγ, and Sα) or AGCTGAGCTG repeats (Sμ and σδ), as revealed by Pustell Matrix dot-plot analysis (MacVector software) (Fig. ).

Techniques: Amplification, Cell Culture, Sequencing, Software, In Vivo, In Vitro, Mutagenesis, Two Tailed Test