Upon activation by antigen, B cells undergo antibody class (isotype) switching, changing from expression of IgM to expression of IgG, IgA or IgE, while maintaining specificity for the same antigen. Since the isotype determines the effector function of the antibody, class switching allows the humoral immune response to adaptively respond to different infectious organisms. Class switching occurs by a DMA recombination event between switch (S) region sequences located upstream of each heavy chain constant (CH) region gene. This process has mechanistic similarities to somatic hypermutation of Ig variable region genes. It has recently become clear that activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by deamination of dC residues within S regions, creating dU residues. It is thought that single strand (ss) DNA nicks are then created by the base excision repair pathway, which converts dU residues to abasic sites, which are then nicked by AP endonuclease. We hypothesize that mismatch repair (MMR) enzymes recognize U:G mismatches created by AID, recruit Exonuclease 1 to the ss breaks created by base excision repair which excises a ss patch resulting in the conversion of these ss breaks to the double-strand breaks (DSBs) that are required for CSR. We also hypothesize that other mismatches and loops formed at S regions due to collapse of R-loops out-of-register will also recruit MMR proteins to S regions during CSR. There are three specific aims which have the goal of providing evidence for these hypotheses.
Aim 1 : To determine the roles of the mismatch proteins in CSR.
Aim 2 : To determine the role of Su tandem repeats and the function of their interaction with MMR proteins.
Aim 3 : To determine the role of uracil-DNA-glycosylase (UNG) in CSR.
In Aim 4, we propose to address the hypothesis that Mlh1 interacts with other mouse MutS homologs during CSR due to our finding that Mlh1 has functions in CSR that are independent of Msh2. Specifically, we propose to investigate whether Msh5 has a role in CSR.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Cellular and Molecular Immunology - B (CMI)
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Macchiarini, Francesca
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University of Massachusetts Medical School Worcester
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Khair, Lyne; Baker, Richard E; Linehan, Erin K et al. (2015) Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells. PLoS Genet 11:e1005438
Kadungure, Tatenda; Ucher, Anna J; Linehan, Erin K et al. (2015) Individual substitution mutations in the AID C terminus that ablate IgH class switch recombination. PLoS One 10:e0134397
Stavnezer, Janet; Linehan, Erin K; Thompson, Mikayla R et al. (2014) Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation. Proc Natl Acad Sci U S A 111:9217-22
Ucher, Anna J; Ranjit, Sanjay; Kadungure, Tatenda et al. (2014) Mismatch repair proteins and AID activity are required for the dominant negative function of C-terminally deleted AID in class switching. J Immunol 193:1440-50
Khair, Lyne; Guikema, Jeroen E J; Linehan, Erin K et al. (2014) ATM increases activation-induced cytidine deaminase activity at downstream S regions during class-switch recombination. J Immunol 192:4887-96
Peng, Min; Xie, Jenny; Ucher, Anna et al. (2014) Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses. EMBO J 33:1698-712
Stavnezer, Janet; Schrader, Carol E (2014) IgH chain class switch recombination: mechanism and regulation. J Immunol 193:5370-8
Vuong, Bao Q; Herrick-Reynolds, Kayleigh; Vaidyanathan, Bharat et al. (2013) A DNA break- and phosphorylation-dependent positive feedback loop promotes immunoglobulin class-switch recombination. Nat Immunol 14:1183-1189
Schrader, Carol E; Linehan, Erin K; Ucher, Anna J et al. (2013) DNA polymerases ? and ? do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations. DNA Repair (Amst) 12:1087-93
Ucher, Anna J; Linehan, Erin K; Teebor, George W et al. (2012) The DNA glycosylases Ogg1 and Nth1 do not contribute to Ig class switching in activated mouse splenic B cells. PLoS One 7:e36061

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