Antibody class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e.g. bacterial lipopolysaccharide (LPS), and results in the expression of different immunoglobulin (Ig) heavy chain constant region (CH) genes without a change in the antibody variable region. Antibody class switching is effected by a DNA recombination event, class switch recombination (CSR), occurring~ between switch (S) sequences. CSR allows antibodies to alter their biological activity while maintaining their antigenic specificity. Humans who are unable to produce IgG have defective immune responses to bacterial and viral pathogens and need to be treated with normal human IgG. The choice of antibody class is regulated by cytokines and different types of B cell activation signals, mostly by regulation of transcription of the unrearranged CH genes. The mechanism of CSR is unknown, except it appears to be initiated by a double-strand DNA break and to be effected by an end-joining recombination event involving error-prone DNA synthesis. During the previous term of this grant, this group has developed a transfectable plasmid that can, undergo CSR specifically in B cells capable of undergoing CSR of their endogenous Ig genes and not in other B cells. This switch plasmid will be used to accomplish two of the Aims proposed here.
Aim #1 is to attempt to clone one or more genes encoding enzymes involved in CSR by cloning cDNAs, which allow the switch plasmid to recombine when transfected into a B cell line incapable of CSR. The function of the protein required for CSR will be determined. Additional genes that interact with this protein will be cloned and studied. This group has recently determined that B cells from mice deficient in the mismatch repair (MMR) proteins, Msh2, Mlhl or Pms2, show a reduction in ability to undergo class switch recombination. Humans and mice lacking these enzymes have a propensity to develop various forms of cancer.
Aim #2 is to examine the role of these MMR enzymes in switch recombination by determining the sequences of S/S junctions in these B cells, examining the hypothesis that MMR proteins are involved in processing ends prior to S/S joining. The group will also identify proteins that interact with MMR proteins in B cells undergoing CSR by the yeast two-hybrid assay and also by co-immunoprecipitation. Proteins that interact with MMR proteins will be tested for their effect on CSR. In addition, mice with deficiencies in proteins already known to interact with MMR proteins, but to remain viable, will be tested for their ability to undergo CSR.
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