DNA Breaks in Class Switch Recombination

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DESCRIPTION (provided by applicant): This proposal is to examine breaks in DNA that occur during the process of immunoglobulin class switch recombination (CSR). CSR is the process by which B lymphocytes exchange the constant region of the immunoglobulin (Ig) molecule they produce in order to most effectively combat the pathogen to which they have been exposed. CSR involves a recombination event in which the IgM constant region gene is deleted and replaced by a downstream constant region gene such as C?. The recombination occurs within DNA located upstream of each constant region gene known as switch (S) regions. As the intervening DNA is excised as a circle, the process requires that double strand breaks are made in both the upstream and downstream S regions. DNA breaks can lead to mutations, translocations and tumors and therefore must be tightly regulated. It is known that activation-induced cytidine deaminase (AID) is required for this process and that it can deaminate cytidines in DNA to generate uracils that can be mutagenic and/or lead to DNA breaks. The experiments proposed here will determine if AID acts directly on Ig S region DNA in vivo to convert cytidines to uracils and also will determine the subsequent steps that lead to break formation. Ligation-mediated (LM)-PCR will be used to detect the exact breakpoint in S region DNA and to test the hypothesis that the breaks instigated by AID activity are initially single-stranded and staggered double strand breaks (DSBs), but that end-processing by DNA repair enzymes can convert some of these breaks to blunt DSBs. The position and structure of breaks will be examined in cells from mice deficient in DNA repair proteins from the mismatch repair (MMR), nucleotide excision repair (NER), and base excision repair (BER) pathways. The BER enzymes UNG and APE can remove uracil from DNA and nick the DNA backbone and are thought to be involved in break formation. Enzymes from the BER pathway will be used to treat genomic DNA from B cells induced to switch in order to detect intermediates in the repair pathway that are predicted by this model.