Somatic hypermutation of immunoglobulin genes occurs at a frequency that is a million times greater than mutation in other genes. Mutations are found in both variable genes and switch regions before constant genes. The molecular mechanism that introduces these mutations is intensely being studied. Hypermutation is initiated when the activation-induced cytidine deaminase (AID) protein deaminates cytosine in DNA to uracil, which causes C:G mutations. However, in B lymphocytes, substitutions of all four bases occur at similar levels, indicating that other proteins are required to generate mutations of A:T base pairs. The MSH2-MSH6 mismatch repair heterodimer and DNA polymerase eta have been implicated in the process, since mice and people deficient for these proteins have fewer mutations of A:T. Using biochemistry and genetics, we are studying how these proteins interact with DNA to generate nucleotide substitutions.

First, we showed that uracil is present on the nontranscribed strand of DNA from bacteria that express AID. Uracils were located primarily at cytosine bases, in accord with the activity of AID on single-strand DNA. This is the first demonstration of dU in cellular DNA, and is an important confirmation of the DNA deamination theory for hypermutation by AID. Second, we determined that MSH2-MSH6 binds to a U:G mismatch, and can enter the pathway at this step. MSH2-MSH6 could then recruit DNA polymerase eta, a low fidelity polymerase, since MSH2 associates with the polymerase in cells. MSH2-MSH6 stimulates the catalytic activity of polymerase eta, allowing it to move faster when it copies nucleotides, so that the polymerase can incorporate mutations at A:T pairs located downstream of the original U:G lesion. This is the first report of a functional interaction between mismatch repair proteins and DNA polymerases. Third, we studied the roles of DNA polymerases eta and iota in mice and humans that lack the enzymes. For polymerase eta, the frequency of hypermutation and class switch recombination was normal, but the types of base changes were different. Polymerase eta-deficient clones had a decrease in mutations at A and T bases, and a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation, and fills in a repair patch downstream of the dU lesion on the nontranscribed strand. For polymerase iota, variable genes were sequenced from mice with the 129 strain defect in the polymerase, and there was no effect on hypermutation or switching. To test for a dual knockout of polymerase eta and other low fidelity polymerases, we will examine mice that are deficient in both enzymes.

A major question in antibody diversity concerns how hypemutation is targeted to the immunoglobulin loci, and is not found elsewhere in DNA from B cells. Mutations are localized to two distinct regions: two kilobases of DNA surrounding and including the rearranged variable gene, and about two kilobases of DNA encompassing the switch region. These regions are downstream of promoters, suggesting that transcription is involved in bringing AID to the locus. To address this question, we will study how transcription proteins interact with the various enzymes involved in the hypermutation pathway. It soon may be possible to assemble all the pieces of the enigmatic hypermutation puzzle.

Martomo SA, Yang WW, Vaisman A, Maas A, Yokoi M, Hoeijmakers JH, Hanaoka F, Woodgate R, Gearhart PJ. (2006) Normal hypermutation in antibody genes from congenic mice defective for DNA polymerase iota. DNA Repair 65:785 [PubMed]

Martomo SA, Gearhart PJ. (2006) Somatic hypermutation: subverted DNA repair. Curr. Opin. Immunol. 161:7040 [PubMed]

Wilson, T.M., Vaisman, A., Martomo, S.A., Sullivan, P., Lan, L., Hanaoka, F., Yasui, A., Woodgate, R., and Gearhart, P.J. (2005) MSH2-MSH6 stimulates DNA polymerase eta?, suggesting a role for A:T mutations in antibody genes. J. Exp. Med. In Press [PubMed]

Mayorov, V.I., Rogozin, I.B., Adkison, L.R., and Gearhart, P.J. (2005) DNA polymerase eta contributes to strand bias of mutations of A versus T in immunoglobulin genes. J. Immunol. 98(3):868-875 [PubMed]

Martomo, S.A., Fu, D., Yang, W.W., Joshi, N.S., and Gearhart, P.J. (2005) Deoxyuridine is generated preferentially in the nontranscribed strand of DNA from cells expressing AID. J. Immunol. 101:67-76 [PubMed]

Martomo, S.A., Yang, W.W., Wersto, R.P., Ohkumo, T., Kondo, Y., Yokoi, M., Masutani, C., Hanaoka, F., and Gearhart, P.J. (2005) Different mutation signatures in DNA polymerase eta- and MSH6-deficient mice suggest separate roles in antibody diversification. Proc. Natl. Acad. Sci. USA 1000(1):57-70 [PubMed]

Seki M, Gearhart PJ, Wood RD. (2005) DNA polymerases and somatic hypermutation of immunoglobulin genes. EMBO Rep. 6:13-24 [PubMed]