Rejoining of DNA Double-Strand Breaks and Genome Stability: From Host-Pathogen Interactions to Break-Induced Mutagensis.

Abstract

An essential task for cell survival is the maintenance of genome stability despite various environmental and physiological stresses. These stresses, such as UV light and ionizing radiation, often damage DNA and result in various types of DNA lesions. If not properly resolved, DNA lesions in humans can cause autoimmune deficiency, neurodegenerative disorders and cancer. Among these lesions, DNA double-strand breaks (DSBs) are one of the most cytotoxic and greatly threaten the integrity of the genome. In addition, DSBs may act as potential hotspots for genomic integration of exogenous DNA fragments, such as the transfer DNA (T-DNA) delivered by the plant pathogen Agrobacterium. DSBs can be repaired by religation of two broken ends by DNA ligase IV via the nonhomologous end joining (NHEJ) pathway, of which the repair fidelity can be compromised by diverse break structures, resulting in mutagenesis. I sought to further understand the complex contribution of NHEJ to genome stability in research projects conducted using both Agrobacterium-mediated plant transformation and Saccharomyces cerevisiae as model systems. My first project examined the process of double-stranded T-DNA formation using functional assays and demonstrated that annealing of synthetic oligonucleotides to the single-stranded T-DNA can initiate such process in plant cells. A second project developed an Agrobacterium-mediated transformation vector system, in which multiple expression cassettes can be assembled on a single vector using zinc finger nucleases (ZFNs) and homing endonucleases, facilitating delivery of multiple genes in plants. A third project investigated the consequences of having a catalytically inactive DNA ligase IV in yeast NHEJ and led to discovery of an imprecise NHEJ pathway mediated by ligase Cdc9. A last project studied the impact of overhang polarity of chromosomal DSBs on the kinetics and fidelity of yeast NHEJ and results suggest that 5’ overhanging DSBs can cause more frequent mutagenesis despite more efficient rejoining as compared to 3’ overhanging DSBs. Collectively, my dissertation research provides new evidence of the mechanisms governing the important process of double-stranded T-DNA formation during plant genetic transformation, as well as new insights into NHEJ mutagenesis which could lead to different human diseases.