Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Bao Q. Vuong

Committee Members

Christine Li

Linda Spatz

Scott Keeney

Jayanta Chaudhuri

Alicia Meléndez

Subject Categories

Biology | Cell Biology | Immunity

Keywords

ATM, MSH2, immunoglobulin, class switch recombination, NHEJ, blunt junctions, synthetic lethality

Abstract

Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain (Igh) gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also function in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, Atm-/- mice were bred to mice deficient for the MMR gene Msh2. Surprisingly, the predicted Mendelian frequencies of Atm-/-Msh2-/- adult mice were not obtained. To generate ATM and MSH2-deficient B cells, Atm was conditionally deleted on an Msh2-/- background using a floxed ATM allele [Atmf] and B cell-specific Cre recombinase expression (CD23-cre) to produce a deleted ATM allele (AtmD). As compared to AtmD/D and Msh2-/- mice and B cells, AtmD/DMsh2-/- mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from AtmD/DMsh2-/- B cells that were induced to switch to IgG1 in vitro showed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, AtmD/D, or Msh2-/- B cells. This data indicates that the absence of both ATM and MSH2 blocks non-homologous end joining (NHEJ), leading to inefficient CSR. We propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID.

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