DNA Repair And Somatic Mutation--Antibody Variable Genes

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Abstract

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. We are studying how mismatch repair proteins and DNA polymerases are involved in the process. First, we showed that uracil is present on the nontranscribed strand of DNA from bacteria that are expressing AID. 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, on a biochemical level, we determined that the MSH2-MSH6 heterodimer binds to a U:G mismatch. Furthermore, MSH2 associates with DNA polymerase eta in cells, and may recruit the heterodimer to U:G mismatches. MSH2-MSH6 then stimulates the catalytic activity of polymerase eta, allowing it to move faster when it copies nucleotides. This biochemical data validates the genetic data that MSH2-MSH6 and polymerase eta work in the same pathway. Third, on a genetic level, we studied the roles of DNA polymerases eta and iota in mice and humans that lack the enzymes. For polymerase eta, variable and switch regions were sequenced from patients with xeroderma pigmentosum variant disease, who are deficient in the polymerase, and from mice that are deficient in the polymerase. The frequency of hypermutation and heavy chain class switching was normal, but the types of base changes were different. Polymerase eta-deficient clones had a decrease in mutations at A and T with a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation. The data indicate that polymerase eta fills in a repair patch downstream of the dU lesion on the nontranscribed strand. For polymerase iota, variable genes will be sequenced from mice with the 129 strain defect in the polymerase. To test for a dual knockout of polymerases eta and iota, we will examine mice that are deficient in both enzymes. The role of ATM, a protein involved in double strand break repair, was also studied in hypermutation and class switch recombination. ATM-deficient mice had normal hypermutation, but defective switching to other isotypes. This indicates that ATM is involved in repairing DNA breaks rather than generating mutations.