Mlh1 can function in antibody class switch recombination independently of Msh2

Abstract

Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes.

Figure 1.

Class switching is similarly reduced in Mlh1- and Msh2-single-deficient and double-deficient mice. Representative FACS^® analysis of splenic B cells induced as indicated for 4d, surface stained with anti-IgM and anti-IgG2a or anti-IgG1 and analyzed by flow cytometry.

Figure 2.

(A) In vitro class switching in splenic B cells from Mlh1- and Msh2-single-deficient and double-deficient mice expressed as percent of switching in B cells from wild-type littermates. Culture conditions for each isotype have been described (reference 16), except for IgG2a which was induced by treatment with LPS + interferon-γ (10 U/ml). The average of three experiments is shown (except n = 2 for Mlh1- IgG1, IgG2a, IgG2b, and IgG3) and error bars represent the SEM. (B) DNA synthesis measured by ³H-thymidine incorporation in 3d cultures with the same inducers used in panel A. Bars show the average of triplicate cultures plus 2 SD, and are representative of two experiments. (C) Cell cycle analysis by propidium iodide staining of cells induced with LPS and anti-δ-dextran for 65 h. The markers estimate the percent of cells in G₀/G₁, S, and G₂/M (left to right) phases of cell cycle.

Figure 3.

Nucleotide sequences surrounding the switch recombination junctions cloned from Mlh1/Msh2-double-deficient cells induced with LPS and anti-δ-dextran. The sequences are of PCR products amplified from 13 independent cultures from two mice. Each cloned sequence (designated M-M) is shown aligned with the sequence of germline Sμ (AF446347, upper sequence) and germline Sγ3, (lower sequence) from 129 × C57BL/6 mice (16), or occasionally from BALB/c mice (MUSIGCD09) as necessary. The boxed nucleotides represent the overlap in sequence between Sμ and Sγ3 with the number of these nucleotides shown to the right of each sequence. A vertical line represents no overlap (no identity). Underlined, boldface nucleotides are designated as insertions as these nucleotides do not appear to be templated by either S region.