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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES013192-09
Application #
8302249
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Shaughnessy, Daniel
Project Start
2004-08-10
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$357,247
Indirect Cost
$136,724
Name
University of Southern California
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Mak, Chi H; Pham, Phuong; Afif, Samir A et al. (2013) A mathematical model for scanning and catalysis on single-stranded DNA, illustrated with activation-induced deoxycytidine deaminase. J Biol Chem 288:29786-95
Jaszczur, Malgorzata; Bertram, Jeffrey G; Pham, Phuong et al. (2013) AID and Apobec3G haphazard deamination and mutational diversity. Cell Mol Life Sci 70:3089-108
Pham, Phuong; Landolph, Alice; Mendez, Carlos et al. (2013) A biochemical analysis linking APOBEC3A to disparate HIV-1 restriction and skin cancer. J Biol Chem 288:29294-304
Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar et al. (2013) GANP interacts with APOBEC3G and facilitates its encapsidation into the virions to reduce HIV-1 infectivity. J Immunol 191:6030-9
Singh, Shailendra Kumar; Maeda, Kazuhiko; Eid, Mohammed Mansour Abbas et al. (2013) GANP regulates recruitment of AID to immunoglobulin variable regions by modulating transcription and nucleosome occupancy. Nat Commun 4:1830
Senavirathne, Gayan; Jaszczur, Malgorzata; Auerbach, Paul A et al. (2012) Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution. J Biol Chem 287:15826-35
Pham, Phuong; Calabrese, Peter; Park, Soo Jung et al. (2011) Analysis of a single-stranded DNA-scanning process in which activation-induced deoxycytidine deaminase (AID) deaminates C to U haphazardly and inefficiently to ensure mutational diversity. J Biol Chem 286:24931-42
Maeda, Kazuhiko; Singh, Shailendra Kumar; Eda, Kazufumi et al. (2010) GANP-mediated recruitment of activation-induced cytidine deaminase to cell nuclei and to immunoglobulin variable region DNA. J Biol Chem 285:23945-53
Chelico, Linda; Pham, Phuong; Petruska, John et al. (2009) Biochemical basis of immunological and retroviral responses to DNA-targeted cytosine deamination by activation-induced cytidine deaminase and APOBEC3G. J Biol Chem 284:27761-5
Chelico, Linda; Pham, Phuong; Goodman, Myron F (2009) Mechanisms of APOBEC3G-catalyzed processive deamination of deoxycytidine on single-stranded DNA. Nat Struct Mol Biol 16:454-5;author reply 455-6

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