Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes.

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

AID: a riddle wrapped in a mystery inside an enigma.

To combat the ever-changing pool of pathogens we face, B cells generate highly optimized antibodies in two distinct steps. A large variety of antibodies are first generated randomly by V(D)J recombination, and then, upon encountering an antigen, antibodies are fine-tuned by somatic hypermutation and class switch recombination--both of which are initiated by the same protein, activation-induced cytidine deaminase (AID). All three processes are highly mutagenic, and mistargeting of each of these has been shown to contribute to tumorigenesis. We study these processes because they provide an excellent model to understand how highly mutagenic reactions are channeled into productive use by cells and the consequent risk this carries. In this review, we will discuss many of the outstanding questions in the field that we grapple with while developing a consistent model for AID action. We will also discuss the complexity added to these models by the recent finding that AID might be part of a demethylase complex.

Replication of N2-ethyldeoxyguanosine DNA adducts in the human embryonic kidney cell line 293.

N(2)-Ethyldeoxyguanosine (N(2)-ethyldGuo) is a DNA adduct formed by reaction of the exocyclic amine of dGuo with the ethanol metabolite acetaldehyde. Because ethanol is a human carcinogen, we assessed the biological consequences of replication of template N(2)-ethyldGuo, in comparison to the well-studied adduct O(6)-ethyldeoxyguanosine (O(6)-ethyldGuo). Single chemically synthesized N(2)-ethyldGuo or O(6)-ethyldGuo adducts were placed site specifically in the suppressor tRNA gene of the mutation reporting shuttle plasmid pLSX. N(2)-EthyldGuo and O(6)-ethyldGuo were both minimally mutagenic in double-stranded pLSX replicated in human 293 cells; however, the placement of deoxyuridines on the complementary strand at 5'- and 3'-positions flanking the adduct resulted in 5- and 22-fold enhancements of the N(2)-ethyldGuo- and O(6)-ethyldGuo-induced mutant fractions, respectively. The fold increase in the N(2)-ethyldGuo-induced mutant fraction in deoxyuridine-containing plasmids was similar after replication in 293T cells, a mismatch repair deficient variant of 293 cells, indicating that postreplication mismatch repair has little role in modulating N(2)-ethyldGuo-mediated mutagenesis. The mutation spectrum generated by N(2)-ethyldGuo consisted primarily of single base deletions and adduct site-targeted transversions, in contrast to the exclusive production of adduct site-targeted transitions by O(6)-ethyldGuo. The yield of progeny plasmids after replication in 293 cells was reduced by the presence of N(2)-ethyldGuo in parental plasmids with or without deoxyuridine to 39 or 19%, respectively. Taken together, these data indicate that N(2)-ethyldGuo in DNA exerts its principal biological activity by blocking translesion DNA synthesis in human cells, resulting in either failure of replication or frameshift deletion mutations.

Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli.

Exocyclic alkylamino purine adducts, including N(2)-ethyldeoxyguanosine, N(2)-isopropyldeoxyguanosine, and N(6)-isopropyldeoxyadenosine, occur as a consequence of reactions of DNA with toxins such as the ethanol metabolite acetaldehyde, diisopropylnitrosamine, and diisopropyltriazene. However, there are few data addressing the biological consequences of these adducts when present in DNA. Therefore, we assessed the mutagenicities of these single, chemically synthesized exocyclic amino adducts when placed site-specifically in the supF gene in the reporter plasmid pLSX and replicated in Escherichia coli, comparing the mutagenic potential of these exocyclic amino adducts to that of O(6)-ethyldeoxyguanosine. Inclusion of deoxyuridines on the strand complementary to the adducts at 5' and 3' flanking positions resulted in mutant fractions of N(2)-ethyldeoxyguanosine and N(2)-isopropyldeoxyguanosine-containing plasmid of 1.4+/-0.5% and 5.7+/-2.5%, respectively, both of which were significantly greater than control plasmid containing deoxyuridines but no adduct (p=0.04 and 0.003, respectively). The mutagenicities of the three exocyclic alkylamino purine adducts tested were of smaller magnitude than O(6)-ethyldeoxyguanosine (mutant fraction=21.2+/-1.2%, p=0.00001) with the N(6)-isopropyldeoxyadenosine being the least mutagenic (mutant fraction=1.2+/-0.5%, p=0.13). The mutation spectrum generated by the N(2)-ethyl and -isopropyldeoxyguanosine adducts included adduct site-targeted G:C-->T:A transversions, adduct site single base deletions, and single base deletions three bases downstream from the adduct, which contrasted sharply with the mutation spectrum generated by the O(6)-ethyldeoxyguanosine lesion of 95% adduct site-targeted transitions. We conclude that N(2)-ethyl and -isopropyldeoxyguanosine are mutagenic adducts in E. coli whose mutation spectra differ markedly from that of O(6)-ethyldeoxyguanosine.