Different mutation signatures in DNA polymerase η- and MSH6-deficient mice suggest separate roles in antibody diversification

Abstract

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 polymerases in adaptive immunity

“…A reduced mutation frequency at A/T bases was observed in these patients 54,55 , an observation that was further confirmed in Polη-deficient mice [56][57][58] (Fig.4).…”

“…Polη-dependent mutations have been observed upstream of switch junctions in human and mouse B cells, but there does not seem to be any quantitative alteration in CSR in the absence of this enzyme [55][56][57]108 . As DNA sequences surrounding switch junctions have no function once CSR has occurred, these mutations are likely to have no physiological role.…”

Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells

“…The dA/dT-biased mutations in the transfected V H 1-DXP′ 1-J H 5 DNA suggest that Msh2, recruited by specific but yet identified features of V H 1-DXP′1-J H 5, triggers MMR in Phase 2. Translesion DNA polymerases, pol η in particular, would be recruited by Msh2 to insert mismatches mostly at dA/dT Indeed, pol η physically interacts with Msh2 (Wilson et al, 2005) and displays a preference in inserting transition at dA/dT (Rogozin et al, 2001;Zeng et al, 2001;Delbos et al, 2005;Martomo et al, 2005), consistent with the predominant dA/dT transitions in the V H 1-DXP′ 1-J H 5 DNA. In turn, the newly generated mismatches at dA/dT could trigger additional rounds of MMR, thereby introducing more mutations at dA/dT residues.…”

Induction of A:T Mutations Is Dependent on Cellular Environment but Independent of Mutation Frequency and Target Gene Location

“…Genetic and biochemical evidence suggests that components of the mismatch repair (MMR) system, including MutS homolog (MSH) 2, MSH6, exonuclease (EXO) 1, and proliferating cell nuclear Ag (PCNA), as well as DNA polymerase (POLH), are required for the induction of A:T mutations (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). It has been proposed that the AID-triggered U:G lesion is recognized by the MSH2/6 heterodimer, followed by EXO-1-mediated strand degradation that generates a gap in the damaged DNA strand.…”