Error bars show mean +/ S.D. in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection Liarozole dihydrochloride of CSR. == INTRODUCTION == Antigen-dependent antibody diversification occurs in two stages: somatic hypermutation (SHM) and class switch recombination (CSR) (1,2). During SHM, antibody-expressing B cell clones that bind antigens are stimulated to proliferate and the variable regions of their immunoglobulin (Ig) genes acquire a large number of mutations. Cell clones expressing mutated immunoglobulins with higher affinities for the antigen undergo positive selection and further rounds of maturation. In contrast to SHM, which yields antibodies with higher affinity through altering the variable region, CSR gives rise to antibodies of a different isotype through an irreversible rearrangement of the heavy chain (CH) locus. The human CHlocus consists of several genes (C, C, Rabbit Polyclonal to CDC2 C3, C1, C1, C2, C4, C and C2), but mature B cells express on their surface initially only IgM or IgD (the latter arising through alternative splicing). During CSR, the antigen/cytokine combination in the environment of the B cell activates transcription of specific switch (S) regions, highly-repetitive sequences flanking the 5 termini of the CHgenes. Transcription then triggers a recombination process during which theIgvariable region is usually joined to one of the downstream constant genes with a concomitant loss of the intervening DNA. Liarozole dihydrochloride In this way, the B-cell switches from expressing the high-avidity IgM to producing high-affinity IgG, IgA or IgE with different biological effector functions (3). SHM and CSR are initiated by activation-induced cytidine deaminase (AID), a protein expressed in antigen-activated B cells (4,5). AID converts numerous cytidines within theIglocus to uracils, in a reaction that is dependent on transcription (6). In the absence of AID, both SHM and CSR are abrogated (7,8). In humans, CSR malfunction causes Hyper-IgM syndrome, characterized by elevated IgM levels and a concomitant decrease or complete absence of IgG, IgA and IgE (9). That SHM and CSR depend not only around the generation of uracils but also on their metabolism was exhibited by the finding that ageing mice lacking uracil-DNA N-glycosylase (UNG), an enzyme that excises uracil from DNA (10), develop B-cell lymphomas (11) and that SHM and CSR are severely attenuated Liarozole dihydrochloride in these animals (12). In humans, recessive mutations in theUNGgene cause Hyper-IgM syndrome (13). Genetic evidence implicated also the mismatch repair (MMR) pathway in these processes: disruption of the mouseMMRgenesMsh2, Msh6, Mlh1, Pms2orExo1led to altered SHM and to a reduction in CSR that ranged from 2- to 7-fold, depending on the gene and theIgserotype (1421). Similarly, patients lacking PMS2 or MSH6 were diagnosed with a profound CSR defect (22,23). These findings were Liarozole dihydrochloride unexpected. Deamination of deoxycytidine, both AID-catalyzed and spontaneous (24), gives rise to U/G mispairs in DNA, however, even though these structures are acknowledged and bound by the human mismatch binding factor MutS (heterodimer of MSH2 and MSH6) (25), they should not be resolved by MMR. Postreplicative MMR has evolved to remove mispaired nucleotides from the newly-synthesized strand during replication. To achieve this goal, MMR proteins need not only detect the mispair, but also direct its repair to the nascent strand. In eukaryotes, this strand is usually distinguished from the template by pre-existing termini, such as gaps between Okazaki fragments, where EXO1 initiates the degradation of the error-containing nascent strand up to and 150 nucleotides past the mispair (26). Because AID-induced U/G mispairs arise in G1 phase of the cell cycle, Liarozole dihydrochloride i.e. in DNA devoid of EXO1 loading sites, they.