DNA damage checkpoints in immunoglobulin diversification and the germinal center reaction

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Checkpoint signalling pathways are instrumental for maintenance of genome integrity in all cells of our body. For targeted genome changes occuring in the adaptive immune system of vertebrates, these pathways need to be modulated in a peculiar manner to allow genetic modification but prevent malignant transformation at the same time. The present proposal assesses the influence of checkpoint signalling on AID-induced immunoglobulin diversification events - somatic hypermutation, class switch recombination and immunoglobulin gene conversion - and the germinal center reaction. In the first funding period, we could show that during secondary immunoglobulin diversification in germinal center B cells, checkpoint signalling by Chk1 and Chk2 plays a role in the regulation of somatic hypermutation and other pathways of secondary immunoglobulin diversification. On the one hand, this occurs via their regulation of error-free DNA repair by homologous recombination, which modulates the error-prone repair pathways of somatic hyypermutation. On the other hand, the common Chk1/2 target protein p53 regulates immunoglobulin mutagenesis in a manner implying differential p53 functionality in two areas of the germinal center: the dark zone where somatic hypermutation occurs, and the light zone where class switch recombination takes place. In the second funding period, we wish to assess the upstream events that affect, as well as the downstream targets that mediate the regulation of immunoglobulin diversification by checkpoint signalling. First, we will investigate how the individual pathways of secondary Ig diversification are linked to cell cycle control, by restricting the activity of AID to individual phases of the cell cycle. Second, we will assess how their activity in different cell cycle phases is modulated by differential regulation of Chk1/2 in the germinal center. Third, we will ask how restriction of homologous recombination at replication forks affects somatic hypermutation. This study will allow important insights into the mechanism and evolution of immunoglobulin diversification, as well as into pathways of its deregulation that lead to the pathogenesis of human B cell lymphoma.