Normal immunoglobulin gene somatic hypermutation in Polκ–Polι double-deficient mice

Shimizu, Takeyuki; Azuma, Takachika; Ishiguro, Masanobu; Kanjo, Naoko; Yamada, Shuichi; Ohmori, Hitoshi

doi:10.1016/j.imlet.2004.11.022

Cited by 31 publications

(14 citation statements)

References 41 publications

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“…We have used GC B cells derived from spleen 2 weeks after immunization with a foreign antigen so that the mutations we observe are induced in a relatively short period by a defined antigen. With this method, the overall mutation frequency in WT mice was consistently ϳ1% and was highly reproducible among different studies (15,17,42,43), allowing us to compare not only the overall mutation frequencies but also the frequency of each type of base substitutions. The current study revealed ϳ40% reduction of the overall mutation frequency in Polh Ϫ/Ϫ mice exclusively because of the greatly reduced A/T mutations.…”

Section: Discussionmentioning

confidence: 92%

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

Masuda

Ouchida

Hara

et al. 2007

Journal of Biological Chemistry

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Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase (POLH) exhibited an ϳ80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in ϳ20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq ؊/؊ Polh ؊/؊ mice, however, did not show a further decrease of A/T mutations as compared with Polh ؊/؊ mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

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Section: Discussionmentioning

confidence: 92%

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

Masuda

Ouchida

Hara

et al. 2007

Journal of Biological Chemistry

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“…POLH is a Y-family low fidelity polymerase, which also includes POLK and POLI [35]. However, neither POLK nor POLI appears to be involved in Ig gene SHM [36][37][38][39]. It remains to be investigated how POLH participates in the SHM of Ig genes and introduces mutations at A:T pairs.…”

Section: Discussionmentioning

confidence: 99%

DNA polymerase η is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation

et al. 2008

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DNA polymerase g (POLH) is required for the generation of A:T mutations during the somatic hypermutation of Ig genes in germinal center B cells. It remains unclear, however, whether POLH is a limiting factor for A:T mutations and how the absence of POLH might affect antibody affinity maturation. We found that the heterozygous Polh 1/À mice exhibited a significant reduction in the frequency of A:T mutations in Ig genes, with each type of base substitutions at a level intermediate between the Polh 1/1 and Polh À/À mice. These observations suggest that Polh is haplo-insufficient for the induction of A:T mutations in Ig genes. Intriguingly, there was also a reduction of C to T and G to A transitions in Polh 1/À mice as compared with WT mice. Polh À/À mice produced decreased serum titers of highaffinity antibodies against a T-dependent antigen, which was associated with a significant reduction in the number of plasma cells secreting high-affinity antibodies. Analysis of the V region revealed that aa substitutions caused by A:T mutations were greatly reduced in Polh À/À mice. These results demonstrate that POLH is a limiting factor for A:T mutations and contributes to the efficient diversification of Ig genes and affinity maturation of antibodies.Key words: Activation-induced cytidine deaminase . Affinity maturation . DNA polymerase g . Immunoglobulin gene . Somatic hypermutation Supporting Information available online IntroductionHigh-affinity antibodies are critical effectors against pathogens and are generated in the germinal center (GC) B cells by the active introduction of mutations at both C:G and A:T pairs in the Ig genes [1]. This hypermutation process is initiated by the activation-induced cytidine deaminase (AID) [2], which is thought to catalyze the deamination of cytosine (C) to uracil (U) on DNA in a transcription-dependent manner [3]. Available genetic evidence suggests that mutations are introduced during replication and the error-prone repair of the AID-triggered U:G mismatch [4][5][6]. Direct replication of the U:G mismatch, or the abasic site formed after excision of U via the uracil DNA glycosylase, could result in mutations at C:G pairs. A number of 2796DNA polymerases, including POLQ and the deoxycytidyl transferase (REV1), have been implicated in generating C:G mutations by replicating over the abasic site [7][8][9][10][11]. The mechanism leading to the mutations at non-damaged A:T pairs still remains largely elusive. The components of the mismatch repair (MMR), including MSH2 and MSH6, and DNA polymerase Z (POLH) are required for the induction of A:T mutations [12][13][14][15][16][17][18][19]. Absence of POLH or MSH2/MSH6 results in 480% reduction of A:T mutations in Ig genes. POLH is a translesion DNA polymerase capable of correctly bypassing UV-induced cyclobutane thymine dimmers and is involved in the suppression of sunlight-induced skin cancers [20,21]. However, POLH is highly inaccurate when replicating undamaged DNA and has an average base substitution error rate that is $1000-fold higher...

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“…DNA polymerase (Pol ) has also been suggested to play a role in SHM in a GC-type Burkitt's lymphoma line (37). However, 129 mice, which lack a functional Pol due to a nonsense mutation in the Poli gene, exhibit a normal frequency and pattern in Ig gene mutations (32,38,39). Deficiency in DNA polymerase (Pol ) has been shown to correlate with dramatically reduced mutations at A͞T pairs in both human and mice (32,33,(40)(41)(42).…”

Section: Discussionmentioning

confidence: 99%

DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

Masuda

Ouchida

Takeuchi

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase (Pol ) has been implicated in mutations at A͞T, but polymerases involved in C͞G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol ) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol ) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J H4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A͞T were unaffected, mutations at C͞G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C͞G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C͞G by replicating over abasic sites generated via uracil-DNA glycosylase.abasic site ͉ low-fidelity DNA polymerase ͉ activation-induced cytidine deaminase ͉ uracil-DNA glycosylase

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Normal immunoglobulin gene somatic hypermutation in Polκ–Polι double-deficient mice

Cited by 31 publications

References 41 publications

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

DNA polymerase η is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation

DNA polymerase θ contributes to the generation of C/G mutations during somatic hypermutation of Ig genes

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