1. Mismatch Recognition and Uracil Excision Provide Complementary Paths to Both Ig Switching and the A/T-Focused Phase of Somatic Mutation 
Cristina Rada, Javier M. Di Noia, Michael S. Neuberger 
Molecular Cell 
Volume 16, Issue 2, Pages (October 2004)
DOI: /j.molcel

2. Figure 1 Paths to Switch Recombination and Somatic Hypermutation
(A) The DNA deamination model for antibody diversification as initially proposed (Petersen-Mahrt et al., 2002) envisaged that AID attacked dC:dG pairs in the vicinity of either the IgV or Ig switch regions generating dU:dG lesions; the dU:dG could then be replicated over, subjected to uracil excision (generating an abasic site), or recognized as a mismatch by MSH2/MSH6. Whereas replication over the dU:dG would lead to transition mutations at dC:dG pairs (phase 1A mutation) and replication over the abasic site would yield both transitions and transversions at dC:dG (phase 1B mutation), it was envisaged that mutations at dA:dT were generated by some form of mutagenic patch repair of the initiating dU:dG lesion (or of derivatives thereof) (phase 2 mutation). The identity of the uracil DNA glycosylase(s) responsible for the uracil excision was originally unspecified, and it was also left open as to which intermediates in the dU:dG base excision repair pathway acted as triggers for phase 2 hypermutation and switch recombination. The pattern of antibody gene diversification in UNG-deficient mice (Rada et al., 2002) revealed that uracil excision by UNG played a dominant role in generating the abasic sites upon which phase 1B mutations were templated (because transversions at dC:dG were obliterated), that UNG played a major, but not wholly essential, role in switch recombination (because switching was substantially diminished, but not blocked), and that UNG was dispensable for phase 2 mutation. Mice singly deficient in MSH2 exhibit a reduction (but not ablation) of phase 2 mutation and a diminished efficiency of switching (Rada et al., 1998).
(B) The antibody diversification phenotype of msh2−/−ung−/− double knockouts indicates that UNG is the only uracil-DNA glycosylase that initiates the major pathway of switch recombination but with MSH2 recognition of the dU:dG providing a backup. Conversely, phase 2 mutation (which was originally envisaged as dA:dT biased but is likely, on the basis of the mutation spectra of the double-knockout mice, to be dA:dT restricted) appears to be triggered by MSH2 recognition but can, at least in the absence of MSH2, be mediated by UNG-recognition of the dU:dG.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
SMUG1 Is Poorly Expressed in Lymphoid Cells, but Enforced Transgenic Overexpression of hSMUG1 Does Not Alleviate the Inhibition of In Vitro Switching in B Cells from ung−/− Mice
(A) Northern blot analysis of endogenous mouse SMUG1 expression in different tissues. Blots were probed for SMUG1 prior to stripping and rehybridizing with a β-actin probe.
(B) Uracil excision activity on a double-stranded oligodeoxyribonucleotide substrate containing a unique uracil residue was monitored in serial 5-fold dilutions of extracts made from control mice splenocytes (left) or LPS-blasted splenic B cells prepared from ung−/− or ung−/−[hSMUG1]-transgenic mice (right). After treatment with endonuclease/NaOH, the 42-mer oligonucleotide substrate (S) is cleaved at the site of uracil excision to create a 26-mer product (P). Where indicated, UNG inhibitor (Ugi) was added to the extracts at a concentration of 0.05 U/μl.
(C) Northern blot analysis of SMUG1 expression in tissues of a pHSE3′-[hSMUG1]-transgenic mouse versus control littermate. The blot was hybridized with a mouse SMUG1 probe, which recognizes both hSMUG1 and mouse SMUG1 transcripts (82% nucleotide identity in the coding region); the two hSMUG1 bands (2.6 and 1.2 Kb) derive from unspliced and spliced hSMUG1 transcripts, respectively, whereas the endogenous mouse SMUG1 transcript is 3.6 Kb.
(D) Flow cytometric profiles of B cells from ung+/+, ung−/−, and ung−/−[hSMUG1] mice that have been cultured for 4 days with LPS+IL4 prior to staining with phycoerythrin-conjugated anti-CD45R(B220) and biotinylated anti-IgG1 with FITC-streptavidin. The percentages of switched sIgG1+ CD45R(B220)+ cells are given in the upper right quadrants.
(E) Histograms depicting (1) the mean percentages of sIgG1+ B cells and (2) the titers of IgG1 in the culture supernatants of splenic B cells from mice of various genotypes that had been cultured for 4 days in the presence of LPS+IL4.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Analysis of B Cells from msh2−/−ung−/− Mice and Their Lack of Switching in Response to LPS
(A) Representative flow cytometric analysis of spleen and Peyer's patch cells from msh2−/−ung−/− and control mice. In the top panels, the percent shown indicates the proportion of germinal center B cells as defined by their CD45R(B220)+ PNAhigh phenotype.
(B) Blasting of purified resting B cells in response to LPS as analyzed by scatter gating after 4 days in culture.
(C) RT-PCR analysis of the induction of sterile (IC) transcripts in control and msh2−/−ung−/−-splenic B cells after 4 days of in vitro culture.
(D) Flow cytometric profiles of splenic B cells from control and knockout mice stained with phycoerythrin-conjugated anti-CD45R(B220) and biotinylated anti-IgG1 with FITC-streptavidin after a 4 day culture with LPS+IL4. The percentage of the scatter-gated cell population that was CD45R(B220)+ sIgG1+ is indicated in the upper right quadrant, with averages from multiple experiments included in Table 1. The abundance of sIgG3+ B cells in LPS-only cultures was 3.4% ± 0.5% in control mice and 0.3% ± 0.1% in double knockouts (not shown).
(E) Titers of IgG1 secreted into the supernatant of LPS+IL4 cultures on day 4 as determined by ELISA.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Lack of Switched Immunoglobulin Isotypes in the Sera of msh2−/−ung−/− Mice
(A) Serum immunoglobulin titers in mice of different genotypes (aged 8–27 weeks) were determined by ELISA. Those from mice younger than 14 weeks are depicted by open circles; those from mice 14 weeks or older by closed circles.
(B) Comparison of the immunoglobulin in sera from matched groups of mice of different ages after affinity purification on protein L-Sepharose, SDS-PAGE separation, and staining with Coomassie.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Complete Lack of Mutation at dA:dT in msh2−/−ung−/− Mice
(A) Extent of mutation accumulation. The sequence of the region flanking the 3′ side of VHJ558DJH4 rearrangements was determined in germinal center B cells sorted from Peyer's patches of 6–7-month-old mice that were either MSH2/UNG deficient (msh2−/−ung−/−) or MSH2/UNG proficient (littermate controls). Pie charts depict the number of unique sequences that carry 0, 1, 2, 3, etc., mutations over the 584 base pair region analyzed. The data from each of two control and each of two msh2−/−ung−/− mice are depicted separately, with the total number of unique sequences analyzed from each mouse indicated in the center of the pie.
(B) Pattern of nucleotide substitutions in the individual control and double-knockout mice. Patterns were deduced either (1) by counting all the nucleotide substitutions identified in the panels of unique sequences or (2) by restricting the database such that for each unique VHDJH rearrangement, only a single mutation of a particular type was scored at an individual nucleotide position. As discussed in the text, the former criterion will overestimate the mutation load in highly clonal datasets, whereas the latter criterion will underestimate it.
(C) Distribution along the intronic VHDJH4 3′ flank of mutations at dC:dG (top panels) and at dA:dT (lower panels). Mutations in control mice are shown above the line and those from msh2−/−ung−/− mice below. The data are taken from the databases in which the stringent criterion has been used to avoid mutation overcounting in dynastically-related sequences. If this process is not applied, the proportion of mutations occurring at major hotspots becomes even more pronounced.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Accumulation of Nucleotide Transitions at dC:dG in the Sμ 5′ Flank of msh2−/− ung−/− and Control Mice
(A) The region flanking the 5′ side of the highly repetitive region of Sμ was PCR amplified (with the same DNA samples from Peyer's patch germinal center B cells used in Figure 5) and sequenced. Pie charts depict the number of sequences that carry 0, 1, 2, 3, etc., mutations over the 579 base pair region analyzed.
(B) The prevalence of sequences that carry insertions/deletions in databases from control and msh2−/− ung−/− mice are compared with histograms. The databases comprise the sequences obtained from both the rearranged VHDJH4 3′ flank and the Sμ 5′ flank. The solid part of the bar indicates the number of sequences that carry mutations other than single nucleotide substitutions. Whereas only a single such sequence (a single-nucleotide deletion) was obtained from the double-knockout mice, small deletions/insertions featured in all databases from individual control mice at a frequency of 5%–10%.
Molecular Cell  , DOI: ( /j.molcel )