Antibody (immunoglobulin, Ig) class switch causes B lymphocytes to switch from producing IgM to producing IgG, IgA or IgE, which improves the ability of the antibody to remove pathogens and bacterial toxins from the body. Class switching occurs by an intrachromosomal DNA recombination event that must be carefully controlled in order to avoid aberrant recombination with other chromosomes (translocations). However, translocations do occur between oncogenes and the IgH locus, and this can lead to B cell lymphomas. During class switching, activation-induced cytidine deaminase (AID) initiates the formation of DNA double strand breaks (DSBs) at switch (S) regions in the Ig heavy chain gene locus (IgH), which are necessary for class switching.
My first Aim i s to determine how deamination of dC's in Ig S regions by AID, forming dU's, results in DSBs. We have shown that AID-induced deamination of dC leads to DNA single-strand breaks (SSBs) via the base excision repair pathway. How these SSBs are then converted to DSBs is less clear. We have reported that another DNA repair pathway, mismatch repair (MMR) is important for this step, and we will investigate its role. We will investigate how SSBs are converted to DSBs by determining the frequency and sites of AID- induced dU's in S regions, how the frequency and positions of AID targets affects frequency of switching, and whether MMR proteins might be recruited to S regions by AID itself.
In Aim 2 we will follow up on our finding during the current term of this grant that AID can instigate DSBs at sites other than the IgH locus in activated B cells. We will determine what makes these other sites targets for AID, and if these DSBs lead to chromosome breaks, deletions and translocations, and whether MMR and other DNA repair proteins known to be involved in CSR, for example, ATM, H2AX, and 53BP1 are involved in making or preventing these DSBs.
The majority of human mature B-cell lymphomas are characterized by reciprocal chromosomal translocations that often involve the immunoglobulin (Ig) genes. The longstanding assumption that these translocations are generated by erroneous antibody class switching, a normal process that is essential for an effective humoral immune response, was verified only recently. Antibody class switching is a process that requires the introduction of DNA breaks into the Ig genes, into special regions called switch regions, and then joining one of these DNA break ends with another DNA break end in the same Ig locus, on the same chromosome. The mechanism of induction of DNA breaks and also how this process is regulated to prevent joining the DNA breaks to another chromosome, which can result in activation of oncogenes and thus B cell lymphomas not understood. The experiments proposed here will determine how the activity of the enzyme AID, which initiates class switching and also chromosomal translocations, is regulated and how it chooses its targets. We have shown that a specific DNA repair pathway in cells, called mismatch repair, which is essential for repairing mistakes during DNA synthesis, is required for formation of most of the DNA breaks during class switching. We seek to further understand its role in converting AID-induced lesions to DNA breaks. Mismatch repair is an essential repair system for eliminating mutations and microsatellite instability, thereby protecting against cellular transformation and malignancy. The hypothesis that this pathway increases DNA breaks seems counterintuitive, but arises directly from our understanding of the role of this pathway during class switching. AID is required for chromosomal translocations between the c-myc oncogene and the Ig locus in mature B-cell plasmacytomas in mouse. Also, many human B-cell lymphomas that originate from germinal center B cells show chromosomal translocations involving the Ig switch regions, suggesting the involvement of class switch processes. IgH translocations often greatly increase oncogene expression due to juxtaposition to the strong IgH enhancers, leading to cellular transformation and malignancy. Whether AID targeting to other loci besides the Ig loci leads to formation of DNA breaks at these sites is, however, unknown. By the use of a genome- wide search method, we have found AID-dependent DNA breaks at several other sites in the genome. We will examine whether these other sites are involved in chromosomal translocations with the Ig locus. We will investigate what makes these other sites targets for AID, and for DNA break formation. Thus our studies will also shed light on the important question of how AID chooses its targets.
|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|
|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|
|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|
|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|
|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|
|Stavnezer, Janet; Schrader, Carol E (2014) IgH chain class switch recombination: mechanism and regulation. J Immunol 193:5370-8|
|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|
|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 14:1183-9|
|Schrader, Carol E; Linehan, Erin K; Ucher, Anna J et al. (2013) DNA polymerases Î² and Î» 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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