2016 Theses Doctoral
RNA:DNA Heteroduplex Resolution in B-Lymphocyte Immunoglobulin Diversification and Genomic Maintenance
Immunoglobulin (Ig) gene diversification plays an essential role in adaptive immunity. Faced with a continuous yet varied stream of self, non-self, and possibly harmful molecules, many organisms have mechanisms in their arsenal that have evolved to match the diversity of the antigens they encounter. In humans and mice, developing B and T lymphocytes go through a first round of genomic alteration — V(D)J recombination — in the bone marrow and the thymus, respectively. B cells can subsequently undergo two additional Ig gene diversification processes in secondary lymphoid tissues. Through somatic hypermutation (SHM), Ig variable regions of stimulated germinal center (GC)-forming B cells are mutated and further diversified, enabling affinity maturation. During class-switch recombination (CSR), on the other hand, B cells in the GC or prior to entering the GC recombine Ig constant regions, swapping the IgM-encoding locus for another isotype constant regions gene (e.g., IgG1, IgG3, IgE, IgA) to allow for different effector functions. Both B cell-specific genomic alterations are initiated when the single-stranded DNA (ssDNA) mutator enzyme activation-induced cytidine deaminase (AID) catalyzes the removal of the amino group off deoxycytidine residues, resulting in deoxyuridines and dU:dG mismatches. Low-fidelity cellular responses to the presence of dU, including the mismatch repair (MMR) and the base-excision repair (BER) pathways, are then thought to introduce mutations in SHM and CSR, as well as cause double-strand breaks (DSBs) repaired through canonical and alternative non-homologous end-joining in CSR.
Though necessary for proper physiological function, these lymphocyte genome diversification processes are rife with danger for B cells and there is strong selective pressure to carefully orchestrate and target them so as not to threaten the genomic integrity of the cells through breaks or other mutations at non-Ig loci. Yet, these events can still occur, as demonstrated by the implication of AID with translocations found in some cancers (e.g., c- MYC:IGH in Burkitt’s lymphoma). Therefore, the mechanisms underlying AID mutagenic activity targeting to physiological deamination substrates have been the focus of several studies.
Protein kinase A (PKA)-dependent phosphorylation of AID at its serine 38 residue has been shown to enable its interaction with replication protein A (RPA) before binding to ssDNA. Others have reported that SPT5 helps target AID to sites of RNA polymerase II (Pol II) stalling, such as the Ig switch sequences. Another cofactor, the RNA exosome complex, helps target the ssDNA mutator AID to both strands of DNA in vivo. The RNA exosome had hitherto been described in the context of RNA processing and degradation as 3’ → 5’ exoribonuclease. Sterile transcript-generating transcription at Ig loci was known to be required for proper AID catalytic activity; the newly described link between the RNA exosome and AID activity raised the prospect that RNA processing, and not mere transcription, might be playing a role in shaping the diversification of the immune repertoire in B lymphocytes.
During CSR, transient three-strand structures called R loops are generated. R loops are formed as the nascent transcript invades the DNA duplex, hybridizing to the template strand, and displacing the non-template one. The G-rich nature of the non-template strand is posited to help stabilize the R loop, which allows the ssDNA mutator AID to use the exposed, non-template strand as a substrate. AID must then access the template strand. Here, we investigate the role that the RNA exosome and a potential cofactor, the putative RNA/DNA helicase senataxin (SETX), play in the sequence of biological events that result in CSR while protecting the cell from R-loop accumulation-associated genomic instability.
- Kazadi_columbia_0054D_13285.pdf binary/octet-stream 25.7 MB Download File
More About This Work
- Academic Units
- Cellular, Molecular and Biomedical Studies
- Thesis Advisors
- Basu, Uttiya
- Ph.D., Columbia University
- Published Here
- May 6, 2016