Molecular Basis of Immunoglobulin Heavy Chain Switch

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DESCRIPTION (provided by applicant): Antibody (immunoglobulin, Ig) class switch causes B lymphocytes to switch from producing IgM to producing IgG, IgA or IgE, which improves the ability of the antibody to remove pathogens and bacterial toxins from the body. Class switching occurs by an intrachromosomal DNA recombination event that must be carefully controlled in order to avoid aberrant recombination with other chromosomes (translocations). However, translocations do occur between oncogenes and the IgH locus, and this can lead to B cell lymphomas. During class switching, activation-induced cytidine deaminase (AID) initiates the formation of DNA double strand breaks (DSBs) at switch (S) regions in the Ig heavy chain gene locus (IgH), which are necessary for class switching. My first Aim is to determine how deamination of dC's in Ig S regions by AID, forming dU's, results in DSBs. We have shown that AID-induced deamination of dC leads to DNA single-strand breaks (SSBs) via the base excision repair pathway. How these SSBs are then converted to DSBs is less clear. We have reported that another DNA repair pathway, mismatch repair (MMR) is important for this step, and we will investigate its role. We will investigate how SSBs are converted to DSBs by determining the frequency and sites of AID- induced dU's in S regions, how the frequency and positions of AID targets affects frequency of switching, and whether MMR proteins might be recruited to S regions by AID itself. In Aim 2 we will follow up on our finding during the current term of this grant that AID can instigate DSBs at sites other than the IgH locus in activated B cells. We will determine what makes these other sites targets for AID, and if these DSBs lead to chromosome breaks, deletions and translocations, and whether MMR and other DNA repair proteins known to be involved in CSR, for example, ATM, H2AX, and 53BP1 are involved in making or preventing these DSBs.