One of the most important human health-related scientific challenges is to understand how the immune system is able to protect against a wide variety of infectious agents. The broad objective of this grant is to investigate the biochemical basis of human immunodiversity. Two key events are required to produce high affinity antibodies from lower specificity antibodies, somatic hypermutation (SHM) and class switch recombination (CSR). Both antibody diversification events require the action of a recently discovered enzyme, activation induced cytidine deaminase (AID). Within the past year it has been shown that AID catalyzes deamination of cytosine to uracil on single-stranded DNA substrates. The deaminations observed in vitro occur most frequently in specific trinucleotide sequences corresponding to SHM hot spot sequences in vivo. Our specific goal, encompassed in four specific aims, is to understand the biochemical basis of SHM. We propose to address this problem first by characterizing the deamination specificity of purified AID in hotspot and coldspot sequences. We will carry out a kinetic analysis to determine binding and catalytic preferences for human wild type AID. We will then investigate AID mutants with altered C deamination specificity that no longer favor hotspot motifs. We will test the hypothesis that wild type AID properties are determined by its ability to scan DNA processively, in contrast to less processive AID mutants exhibiting reduced deamination specificity. Only actively transcribed immunoglobulin variable genes undergo SHM. In the third specific aim, we will investigate AID-catalyzed deamination using a eukaryotic transcription system, including RNA polymerase II and transcription factors. Using """"""""classical"""""""" biochemical add-back experiments, we will perform a systematic search for transcription components that function to target AID to the transcription apparatus. The first three specific aims are intended to clarify the role of AID in initiating C to T transition mutations in the non-transcribed stand, at an important class of hotspot motifs in SHM. A longer-term goal, embodied in specific aim four, is to investigate mutational strand specificity, including mutations at A and T sequences.
This aim will investigate AID in conjunction with base excision repair, mismatch repair and recently discovered error-prone DNA polymerases. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES013192-01
Application #
6810441
Study Section
Special Emphasis Panel (ZRG1-CDF-2 (90))
Program Officer
Tinkle, Sally S
Project Start
2004-08-10
Project End
2009-07-31
Budget Start
2004-08-10
Budget End
2005-07-31
Support Year
1
Fiscal Year
2004
Total Cost
$385,938
Indirect Cost
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
Pham, Phuong; Afif, Samir A; Shimoda, Mayuko et al. (2017) Activation-induced deoxycytidine deaminase: Structural basis for favoring WRC hot motif specificities unique among APOBEC family members. DNA Repair (Amst) 54:8-12
Pham, Phuong; Afif, Samir A; Shimoda, Mayuko et al. (2016) Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification. DNA Repair (Amst) 43:48-56
Goodman, Myron F (2016) Better living with hyper-mutation. Environ Mol Mutagen 57:421-34
Senavirathne, Gayan; Bertram, Jeffrey G; Jaszczur, Malgorzata et al. (2015) Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution. Nat Commun 6:10209
Mak, Chi H; Pham, Phuong; Afif, Samir A et al. (2015) Random-walk enzymes. Phys Rev E Stat Nonlin Soft Matter Phys 92:032717
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
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 191:6030-6039
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
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

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