Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions

Abstract

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1(-)(/)(-) splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Smicro segments and recombined Smicro-Sgamma3 segments cloned from Ercc1(-)(/)(-) splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1(-)(/)(-) cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S-S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.

Figure 1.

Reduced switching in Ercc1 ^−/− splenic B cells is not due to a reduction in cell proliferation. (A) The percent of Ercc1 ^−/− B cells in S phase, as determined by flow cytometry of propidium iodide–stained cells, after stimulation with LPS plus anti–δ dextran for 2 d is slightly reduced. One heterozygous and two WT littermates are shown for comparison. (B) CFSE-stained B cells cultured for 4 d with the indicated inducers lose CFSE staining by 50% with each cell division. The dark line represents the fluorescence tracing and the peaks within the fluorescence tracing are integrations representing individual cell generations that are enumerated above the graph. (C) Cells from the indicated generations were gated on and analyzed for IgG1 expression (other isotypes not shown). (D) Surface IgG or IgA expression by Ercc1 ^{− / −} cells is shown as a percent of expression of WT cells (+ SEM) after 4 d in culture with appropriate CSR inducers. Only cells that divided six or more times were analyzed and the percent switching to each isotype relative to WT is shown (+ SEM). n = 3 for all isotypes, except for IgA, where n = 1. The differences between WT and Ercc1 ^{− / −} switching for all isotypes are highly significant: P < 0.001 as determined by a paired t test.

Figure 2.

Mutations are more clustered at the Sμ–Sγ3 junctions in (A) WT B cells than in (B) Ercc1 ^{− / −} B cells. The mutation density per 50-bp segment is plotted. Only segments in which the entire sequence was available are included. The significance of the differences in mutation distribution on both the Sμ and Sγ3 sides, and also in toto, are all P < 0.001; two-tailed Mann-Whitney test.