Volume 19, Issue 4, Pages 515-524 (October 2003) The Sμ Tandem Repeat Region Is Critical for Ig Isotype Switching in the Absence of Msh2  Irene M. Min, Carol E. Schrader, Joycelyn Vardo, Thomas M. Luby, Nicole D'Avirro, Janet Stavnezer, Erik Selsing  Immunity  Volume 19, Issue 4, Pages 515-524 (October 2003) DOI: 10.1016/S1074-7613(03)00262-0

Figure 1 Isotype Switching to IgG1 Is Greatly Reduced in Sμ−/−:msh2−/− Mice Splenic B cells from age-matched mutant mice and control wild-type mice were cultured for 4 days with LPS and IL4 to stimulate IgG1 class switching. Flow cytometric analyses of IgG1-expressing B cells are shown; the percentages of IgG1-positive cells are indicated in each diagram. Note that wild-type cells treated with LPS alone did not switch to IgG1. Immunity 2003 19, 515-524DOI: (10.1016/S1074-7613(03)00262-0)

Figure 2 Reductions of Isotype Switching for All of the Major Isotypes in Sμ−/−:msh2−/− Mice As in Figure 1, flow cytometry was used to estimate the efficiency of isotype switching with in vitro stimulated B cells. The bar graphs represent the average switching frequencies and the corresponding standard error of means (SEM) in three to six independent experiments for the four mouse genotypes indicated. For each isotype, values for wild-type mice are set to 100%; values for each mutant genotype are then calculated relative to wild-type. For each isotype, the average frequencies of switched cells in the stimulated wild-type cultures were IgG1, 28%; IgG2a, 13.0%; IgG2b, 13.2%; IgG3, 6.3%; IgA, 4.0%. Immunity 2003 19, 515-524DOI: (10.1016/S1074-7613(03)00262-0)

Figure 3 DC-PCR Analyses Show that Both IgG1 and IgG3 Class Switch DNA Recombinations Are Substantially Reduced in Sμ−/−:msh2−/− Mice (A) Genomic DNAs from IgG1 and IgG3 switched cells were prepared and 2-fold dilutions were subjected to Sμ/Sγ1 and Sμ/Sγ3 DC-PCR, respectively. Samples were normalized using control nicotinic acetylcholine receptor (nAChR) DC-PCR reactions. (B) Average values for the levels of Sμ/Sγ1 and Sμ/Sγ3 switch recombinations in mutant mice relative to wild-type mice. Values for wild-type mice are set to 100%. Immunity 2003 19, 515-524DOI: (10.1016/S1074-7613(03)00262-0)

Figure 4 Switch Recombination Sites from Sμ−/− Mice Do Not Focus on Hairpin Structures in the JH-Cμ Intron Two maps locating representative Sμ/Sγ1 and Sμ/Sγ3 junctions relative to predicted stem-loop structures of the single-stranded Sμ region are shown. Arrowheads indicate switch junction sites that have no shared microhomologies whereas junctions that exhibit shared microhomologies are marked as boxes. Switch sites that are derived from Sμ−/− mice are indicated by open arrowheads or open boxes. Switch sites from Sμ−/−:msh2−/− mice are marked with solid arrowheads or solid boxes. The sequence in (A) spans the HindIII site located immediately downstream of the loxP site in the Sμ− allele (Luby et al., 2001) and this HindIII sequence corresponds to nucleotide 1207 in Sμ genomic sequence from GenBank accession number J00442. This HindIII site is present within the loop of the first hairpin on the left. The 5′ end of the sequence in (B) is located 314 bp downstream of the HindIII site in (A). Immunity 2003 19, 515-524DOI: (10.1016/S1074-7613(03)00262-0)

Figure 5 Model for Msh2 Action in SμTR Region and Sequences Surrounding the SμTR during Class Switch DNA Recombination The top diagram shows the Sμ tandem repeat region together with DNA sequences flanking SμTR. Sites of single-stranded class switch DNA cleavages are indicated by arrows. DNA cleavages within SμTR are proposed to be closely spaced and, therefore, predominantly generate DNA ends that have short single-stranded extensions. These can be joined with downstream S region DNA ends by DNA-PK/Ku complex-mediated nonhomologous end-joining (NHEJ) pathway independent of Msh2 (left panel). In contrast, sequences outside of the SμTR region (right panel) have more widely spaced cleavage sites, which can lead to flap DNA ends with longer 5′ or 3′ extensions. Msh2 is suggested to recruit nucleases to end-process the 3′ flap DNA ends before they are recombined by the NHEJ mechanism. Rectangles and ovals represent the DNA-PK/Ku complex and Msh2 complex, respectively. Immunity 2003 19, 515-524DOI: (10.1016/S1074-7613(03)00262-0)