Year: 2020
Type: dissertation
Abstract: To meet the depth and breadth of antigen space brought to bear by potential pathogens, adaptive immunity requires somatic diversification of antigen receptors through programmed DNA damage and concomitant recombination and repair of antigen receptor loci. This biological fact is conserved in all vertebrate lineages and is elaborated upon with secondary phenomena such as class switch recombination (CSR). Together, these recombinatorial adaptive immune processes are recapitulated billions of times over in each organism, mostly without negative consequences such as undesirable mutagenesis and genomic instability. This juxtaposition of damage and repair in a physiological model has been felicitous to the understanding of DNA repair more generally, and insights into the basis of adaptive immunity and DNA repair have played off together symbiotically. In that tradition, this manuscript endeavours to examine the molecular requirements of class switch recombination (CSR) along two complementary axes. Firstly, although it is known that double-stranded breaks (DSB) are substrates necessary for CSR, it is unknown how the structure and polarity of DSBs affects downstream DNA repair. Using the Cas9 enzyme and related variants, I generated DSBs of defined structures near the switch regions of the immunoglobulin heavy chain locus (Igh) and examined the resultant CSR frequency and repair junction characteristics. I found that single-stranded breaks upwards of 250 bp apart on opposite strands could resolve as DSBs in CSR. Moreover, 5′ DSBs were more preferred CSR substrates than 3′ DSBs, although both types of staggered DSBs had a higher level of resection and microhomology usage at the repair junction than blunt DSBs. Taken together, these observations suggest that DSB structure influences the type of repair effected as well as the frequency of productive CSR. I also investigated the effect of the newly-identified shieldin complex downstream of 53BP1 on concluding CSR. Shld2–/– B cells have profoundly impaired CSR, although B cell development and V(D)J recombination were not grossly affected. Additionally, I observed an increased Ig-low population in shieldin-deficient switching B cells that was the result of extreme resection impairing Ig expression, rather than increased inversional recombination, and congruent with the proposed function of shieldin.
Source: TSpace (University of Toronto)
Author: Alexanda K. Ling
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3161047878