Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6 −/− clones, whereas mutations were dispersed in Msh3 −/− and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.

Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA.

Hypermutation in immunoglobulin genes produces a high frequency of substitutions of all four bases, which are likely generated by low-fidelity DNA polymerases. Indeed, humans deficient for DNA polymerase (pol) have decreased substitutions of A⅐T base pairs in variable and switch regions. To study the role of pol in a genetically tractable system, we created mice lacking pol . B cells from Polh ؊/؊ mice produced normal amounts of IgG, indicating that pol does not affect class switch recombination. Similar to their human counterparts, variable and switch regions from Polh ؊/؊ mice had fewer substitutions of A⅐T base pairs and correspondingly more mutations of C⅐G base pairs, which firmly establishes a central role for pol in hypermutation. Notably, the location and types of substitutions differ markedly from those in Msh6 ؊/؊ clones, which also have fewer A⅐T mutations. The data suggest that pol preferentially synthesizes a repair patch on the nontranscribed strand, whereas MSH6 functions to generate the patch.class switch recombination ͉ somatic hypermutation ͉ low-fidelity DNA polymerase ͉ mismatch repair I mmunoglobulin (Ig) genes undergo a high frequency of somatic hypermutation during B cell activation. The mutations, which are predominantly nucleotide substitutions, are introduced into Ϸ2-kb regions of DNA containing variable (V) region genes and switch (S) regions that precede each constant region gene. Mutations in V gene exons can generate high-affinity antibodies that bind to antigen, whereas mutations in S region introns are linked to class switch recombination that produces different isotypes (reviewed in refs. 1-3). Hypermutation and switching are both initiated by the activation-induced cytidine deaminase protein (4, 5), which deaminates dC to dU in DNA (6-8). The dU lesion likely generates mutation in two phases, as originally proposed by Neuberger and colleagues (6). In phase 1, the dU lesion could generate mutations of C bases, or of G bases if C is deaminated on the complementary strand. For example, dU could remain in the DNA and be copied by a high-fidelity DNA polymerase (pol) to produce transitions of C⅐G. Alternatively, dU could be removed by uracil DNA glycosylase, and the abasic site could be copied or repaired by a low-fidelity pol to produce transitions and transversions of C⅐G.In phase 2, the dU lesion could initiate a repair patch and generate mutations of neighboring bases, including A and T. In this pathway, the MSH2-MSH6 heterodimer (9-11) could bind to the U⅐G mispair (12, 13), an endonuclease could nick the DNA, and exonuclease 1 (14) would create a gap near the original dU lesion. The gap would then be filled in by a low-fidelity pol, and the types of substitutions would reflect the specificity of the pol. A plethora of pols have been studied for their role in hypermutation in animals and cells:

DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.

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