A fundamental health-related scientific challenge addresses how the immune system protects against a wide variety of infectious agents. Our broad objective is to investigate the biochemical basis of human immunodiversity. Two key events are required to produce high affinity antibodies (Ab) from lower specificity antibodies, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Ab diversification requires the action of a B-cell specific enzyme, activation induced cytidine deaminase (AID). AID, a member of the APOBEC family of nucleic acid cytidine deaminases, converts C?U during transcription of immunoglobulin genes to initiate SHM and CSR. APOBEC3G (A3G), which converts C?U on retroviral cDNA, plays an instrumental role in restricting infection of the AIDS virus (HIV-1) in T cells. From a biological perspective, an understanding of the biochemical properties of AID and A3G is essential to grasp the programmed roles for these enzymes in ensuring Ab diversification and in imposing innate resistance against retroviral infection. From a mechanistic perspective, an understanding of the biochemical properties of AID and A3G entail deciphering the stochastic properties of processive enzymes designed to deaminate C bases in DNA strands. AID- and A3G-catalyzed deaminations occur in a "haphazard" manner resulting in diverse mutations distributed throughout their DNA targets, Ig variable and switch regions for AID, and HIV-1 cDNA for A3G. An in-depth in vitro analysis aimed at revealing the biochemical basis for the diverse distribution of mutations is a key objective of this proposal. Both enzymes employ a processive scanning process, involving sliding and jumping along ssDNA. Specific Aims 1 and 3 analyze scanning and deamination mechanisms for AID and A3G, respectively. Specific Aim 2 examines the deamination properties of WT AID compared to AID mutants associated with hyper-IgM-2 syndrome in humans, in which Ab diversification fails to occur. Specific Aim 4 uses laser single molecule microscopy to visualize scanning by AID and A3G and to test 3-D scanning mechanisms derived from Aims 1-3. AID instigates a cascade of mutational events involving error-prone DNA polymerases, base excision repair (BER) and mismatch repair (MMR) enzymes culminating in a pool of highly mutated antibody genes. In Specific Aim 5, we broaden our perspective and look "downstream" from AID, to investigate in vitro systems for error-prone mismatch repair and base excision repair. PUBLIC HEALTH REVELANCE: In all organisms, from microorganisms to humans, it is axiomatic that mutations are almost always deleterious, serving as a fundamental cause of numerous diseases, most prominently cancer. There are, however, programmed pathways involving "error-prone" DNA repair that deliberately introduce mutations at extremely high levels. These mutational pathways are beneficial, and often essential in providing immunological diversity, general fitness and avoidance of cell death. The proposed research explores the mechanisms used by two human DNA cytidine deaminases, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G). AID ensures antibody diversification. A3G imposes innate resistance against HIV-1 retroviral infection. The enzymes are under tight regulation, because cancer is known to occur if AID or A3G are expressed at the wrong time or in the wrong place. The research entails deciphering the biochemical properties of AID- and A3G-catalyzed deaminations, which occur in a "haphazard" manner resulting in diverse mutations distributed throughout their DNA targets, immunoglobulin variable regions for AID, and HIV-1 complementary DNA for A3G.