High affinity antibody responses require secondary immunoglobulin gene (Ig) diversification by somatic hypermutation (SHM) and class switch recombination (CSR) in germinal centers. Both of these reactions are mediated by activation induced cytidine deaminase (AID), an enzyme that deaminates cytosine residues in single-stranded DNA (ssDNA) exposed by stalled RNA polymerase II during transcription (1-3). Although AID has a strong preference for targeting Ig genes, it also mutates a large number of non-Ig loci. AID induced U:G mismatches are processed by one of several repair pathways to yield non-templated mutations or DSBs, which are obligate intermediates in CSR that are normally recognized by DNA damage response proteins and repaired by non-homologous end joining (NHEJ). However, these DSBs can also serve as substrates for chromosome translocations (4-6). 53BP1 is a DNA damage response protein that is recruited to DNA double strand breaks (DSBs) and is essential for their efficient repair. We, and others have shown that 53BP1 contributes to DNA repair during CSR by preventing end resection and by bringing together DSBs separated by as much as 200kb (7-13). 53BP1 is a large protein that is implicated in the etiology of malignancy and that contains a number of domains that are essential to its function in CSR (7, 8). Most importantly, the N terminal domain of 53BP1 is a target for phosphorylation by ataxia telangiectasia mutated (ATM) and is essential for both end protection and synapsis (7, 8), but its function is not known. We have discovered that phosphorylated 53BP1 acts as an adaptor for Rif1 and that this interaction is required for DNA end protection and CSR. The long-term goals of the proposed research are to define the genes that are damaged by AID in germinal center B cells, understand the mechanism of AID targeting, and obtain a molecular understanding of how Rif1 contributes to CSR. To accomplish these goals we propose three specific aims. First, we will define the genes that are damaged by AID and that serve as substrates for translocation in GC B cells in chronic infection using malaria as a model. Second, we will define the mechanism of AID targeting and the role of Spt5. Third, we will define the mechanism by which Rif1 contributes to CSR. Taken together, these experiments should provide an understanding of antibody gene diversification reactions and how they contribute to the genesis of B cell malignancy.

Public Health Relevance

High affinity, AID dependent, antibody responses are essential to nearly all vaccines. The proposed research aims to develop an understanding of how antibodies are modified and repaired after AID mediated DNA damage with the long-term goal of being able to impact vaccine development and prevent the off target effects of AID that are associated with malignancy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R37AI037526-20A1
Application #
8694774
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Nasseri, M Faraz
Project Start
1994-09-01
Project End
2019-01-31
Budget Start
2014-02-15
Budget End
2015-01-31
Support Year
20
Fiscal Year
2014
Total Cost
$381,375
Indirect Cost
$156,375
Name
Rockefeller University
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Wang, Qiao; Kieffer-Kwon, Kyong-Rim; Oliveira, Thiago Y et al. (2017) The cell cycle restricts activation-induced cytidine deaminase activity to early G1. J Exp Med 214:49-58
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Dong, Junchao; Panchakshari, Rohit A; Zhang, Tingting et al. (2015) Orientation-specific joining of AID-initiated DNA breaks promotes antibody class switching. Nature 525:134-139
Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy et al. (2015) An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex. J Allergy Clin Immunol 135:998-1007.e6
Robbiani, Davide F; Deroubaix, Stephanie; Feldhahn, Niklas et al. (2015) Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma. Cell 162:727-37

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