Somatic hypermutation introduces single base changes into rearranged and expressed, altering DNA sequence in order to increase the affinity of the immune response or the diversity of the immune repertoire. In the past few years, there has been extraordinary progress toward understanding the hypermutation mechanism. We now know that hypermutation is initiated by the DNA deaminase, Activation-Induced Deaminase (AID), which converts C to U in transcribed V regions. This lesion is then processed by ubiquitous repair factors which, paradoxically, promote mutagenic rather than faithful repair at the Ig loci. The long-term goal of our research is to define the mechanisms of V region mutagenesis. We propose three specific aims to reach that goal. (1) We will establish how DNA cleavage and ligation occur at diversifying Ig genes, and determine how the function of the MRE11 AP lyase activity in Ig gene diversification reflects its highly conserved role in DNA repair. (2) We will determine how spatiotemporal regulation of positioning of the Ig genes within the nucleus contributes to activation and targeting of diversification, and identify the mechanisms that direct Ig gene positioning. (3) We will ask how the activity of AID is specifically targeted to the Ig genes, while the vast majority of the genome is protected from AID attack. The proposed experiments will not only solve a fascinating puzzle in basic biology, they also have important implications for our understanding of human health and disease. This research will identify molecular targets which can be manipulated to enhance the efficiency of immunization, and provide new understanding about the factors and pathways that maintain genomic stability in normal cells and destabilize the genome in cancer.