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

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.PHDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111334/1/zbliang_1.pd

Spacelike hypersurfaces with negative total energy in de Sitter spacetime

De Sitter spacetime can be separated into two parts along two kinds of hypersurfaces and the half-de Sitter spacetimes are covered by the planar and hyperbolic coordinates respectively. Two positive energy theorems were proved previously for certain $\P$-asymptotically de Sitter and \H-asymptotically de Sitter initial data sets by the second author and collaborators. These initial data sets are asymptotic to time slices of the two kinds of half-de Sitter spacetimes respectively, and their mean curvatures are bounded from above by certain constants. While the mean curvatures violate these conditions, the spacelike hypersurfaces with negative total energy in the two kinds of half-de Sitter spacetimes are constructed in this short paper.Comment: 11 pages, final version, to appear in J. Math. Phy

Direct Electrochemistry of Glucose Oxidase at a Gold Electrode Modified with Single-Wall Carbon Nanotubes

The direct electrochemistry of glucose oxidase (GOD) was accomplished at a gold electrode modified with single-wall carbon nanotubes (SWNTs). A pair of welldefined redox peaks was obtained for GOD with the reduction peak potential at –0.465 V and a peak potential separation of 23 mV at pH 7.0. Both FT-IR spectra and the dependence of the reduction peak current on the scan rate revealed that GOD adsorbed onto the SWNT surfaces. The redox wave corresponds to the redox center of the flavin adenine dinucleotide(FAD) of the GOD adsorbate. The electron transfer rate of GOD redox reaction was greatly enhanced at the SWNT-modified electrode. The peak potential was shown to be pH dependent. Verified by spectral methods, the specific enzyme activity of GOD adsorbates at the SWNTs appears to be retained

Direct electrochemistry of glucose oxidase at a gold electrode modified with single-wall carbon nanotubes

Abstract: The direct electrochemistry of glucose oxidase (GOD) was accomplished at a gold electrode modified with single-wall carbon nanotubes (SWNTs). A pair of welldefined redox peaks was obtained for GOD with the reduction peak potential at –0.465 V and a peak potential separation of 23 mV at pH 7.0. Both FT-IR spectra and the dependence of the reduction peak current on the scan rate revealed that GOD adsorbed onto the SWNT surfaces. The redox wave corresponds to the redox center of the flavin adenine dinucleotide(FAD) of the GOD adsorbate. The electron transfer rate of GOD redox reaction was greatly enhanced at the SWNT-modified electrode. The peak potential was shown to be pH dependent. Verified by spectral methods, the specific enzyme activity of GOD adsorbates at the SWNTs appears to be retained

Zinc Finger Nuclease and Homing Endonuclease-Mediated Assembly of Multigene Plant Transformation Vectors1[OA]

Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids

Enhanced eMAGE applied to identify genetic factors of nuclear hormone receptor dysfunction via combinatorial gene editing

Abstract Technologies that generate precise combinatorial genome modifications are well suited to dissect the polygenic basis of complex phenotypes and engineer synthetic genomes. Genome modifications with engineered nucleases can lead to undesirable repair outcomes through imprecise homology-directed repair, requiring non-cleavable gene editing strategies. Eukaryotic multiplex genome engineering (eMAGE) generates precise combinatorial genome modifications in Saccharomyces cerevisiae without generating DNA breaks or using engineered nucleases. Here, we systematically optimize eMAGE to achieve 90% editing frequency, reduce workflow time, and extend editing distance to 20 kb. We further engineer an inducible dominant negative mismatch repair system, allowing for high-efficiency editing via eMAGE while suppressing the elevated background mutation rate 17-fold resulting from mismatch repair inactivation. We apply these advances to construct a library of cancer-associated mutations in the ligand-binding domains of human estrogen receptor alpha and progesterone receptor to understand their impact on ligand-independent autoactivation. We validate that this yeast model captures autoactivation mutations characterized in human breast cancer models and further leads to the discovery of several previously uncharacterized autoactivating mutations. This work demonstrates the development and optimization of a cleavage-free method of genome editing well suited for applications requiring efficient multiplex editing with minimal background mutations

<i>Saccharomyces cerevisiae</i> DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

<div><p>DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation <i>in vitro</i> and NHEJ activity <i>in vivo</i>, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that <i>dnl4</i>-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as <i>dnl4</i> point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.</p></div