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

Rika Ouchida,Jiyang O-Wang,Tomohiro Kurosaki,Fumio Hanaoka,Keiji Masuda,Masaki Hikida,Richard D. Wood,Masayuki Yokoi,Mineaki Seki,Chikahide Masutani

doi:10.1074/jbc.m611849200

Abstract

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 eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% 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.

Highlights

The immunoglobulin genes are assembled in developing B cells by recombination-activating gene-mediated rearrangement of the germline V, D, and J gene segments [1,2,3]
Normal B and T Cell Development and Maturation in PolqϪ/Ϫ PolhϪ/Ϫ Mice—FACS analysis of the bone marrow cells of WT, PolqϪ/Ϫ, PolhϪ/Ϫ, and PolqϪ/ϪPolhϪ/Ϫ mice revealed no obvious differences in the percentages of CD43ϩ IgMϪ progenitor, CD43ϪIgMϪ precursor, and CD43ϪIgMϩ B cells after gating on the B220ϩ cells (Fig. 1A)
Normal B Cell Responses and Class Switch Recombination in PolqϪ/ϪPolhϪ/Ϫ Mice—Having confirmed that B and T cell development was phenotypically indistinguishable among the different mice, we analyzed B cell function as assessed by proliferative responses to various activation signals

Summary

Introduction

The immunoglobulin genes are assembled in developing B cells by recombination-activating gene-mediated rearrangement of the germline V, D, and J gene segments [1,2,3]. Polq؊/؊Polh؊/؊ mice, 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.

Results

Conclusion