Three phenotypically distinct processes-somatic hypermutation, gene conversion, and switch recombination-remodel the functionally rearranged immunoglobulin (Ig) loci in B cells. Somatic hypermutation and switch recombination have recently been shown to depend on the activation-induced deaminase (AID) gene product. Here, we show that the disruption of the AID gene in the chicken B cell line DT40 completely blocks Ig gene conversion and that this block can be complemented by reintroduction of the AID complementary DNA. This demonstrates that the AID master gene controls all B cell-specific modifications of vertebrate Ig genes.

Pax5 is indispensable for the commitment of early lymphoid progenitors to the B cell lineage as well as for the development of B cells. To better understand the functional importance of Pax5 at the later stages of B cell differentiation, we established a Pax5-deficient DT40 B cell line. The Pax5(-/-) cells exhibited slower growth, decreased surface IgM expression, and total loss of B cell receptor signaling. Moreover, the expression of the plasma cell-characteristic transcription factors Blimp-1 and XBP-1 were significantly upregulated and the expression of Bcl-6 diminished in the Pax5(-/-) cells, and this alteration was normalized by restored Pax5 expression. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results indicate that downregulation of Pax5 function promotes the plasma cell differentiation of B cells.

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Depending on the species and the lymphoid organ, activation-induced cytidine deaminase (AID) expression triggers diversification of the rearranged immunoglobulin (Ig) genes by pseudo V (ψV) gene- templated gene conversion or somatic hypermutation. To investigate how AID can alternatively induce recombination or hypermutation, ψV gene deletions were introduced into the rearranged light chain locus of the DT40 B-cell line. We show that the stepwise removal of the ψV donors not only reduces and eventually abolishes Ig gene conversion, but also activates AID-dependent Ig hypermutation. This strongly supports a model in which AID induces a common modification in the rearranged V(D)J segment, leading to a conversion tract in the presence of nearby donor sequences and to a point mutation in their absence.

Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.DNA double-strand breaks (DSBs) are among a plethora of lesions that threaten the integrity of the genome. If not properly processed, DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements such as translocations, inversions, or deletions. These rearrangements can contribute to cell dysfunction, cell death, or carcinogenesis (22). DSBs can arise through the action of exogenous DNA-damaging agents, but they also arise from endogenous sources, such as oxidative DNA damage and as a consequence of DNA replication (10,22). Homologous recombination is a major DNA repair pathway by which DSBs are repaired. Homologous recombination is generally a precise way of resolving DSBs, because it uses homologous sequence, usually provided on the sister chromatid, as a repair template (54).Homologous recombination is a complex process requiring a number of proteins of the RAD52 epistasis group, including Rad51 and Rad54. Rad51 is the key player in this process because it is critical for homology recognition and performs strand exchange between recombining DNA molecules. A pivotal intermediate in these reactions is the Rad51 nucleoprotein filament. This forms when Rad51 polymerizes on singlestranded DNA that results from DNA damage processing (54). Rad54 is an important accessory factor for Rad51 (56). A number of biochemical characteristics of Rad54 have been well defined for different species ranging from yeasts to humans (8,18,24,31,37,38,42,47,48,53,55,59). Rad54 is a doublestranded-DNA-dependent ATPase that can translocate on DNA, thereby affecting DNA topology. Biochemically, Rad54 has been implicated in participation in multiple steps of homologous recombination. It can stabilize the Rad51 nucleoprotein filament in an early stage of recombination (30). At a subsequent stage it can promote chromatin rem...