Small molecules have played an important role in defining the functions and identities of numerous proteins involved in fundamental biological processes as well as pathways involved in disease. Chemical genetics represents the formalization of this process into a defined field desiring to achieve the breadth and specificity of classical genetics. In order to gain full advantage of a small molecule's ability to perturb the cell for novel or desired phenotypes, a complete understanding of the molecule's mechanism of action must be achieved. Identification of the biological targets of a molecule represents the most direct approach to attaining this knowledge. In our strategy to find novel mechanisms to target cancers with oncogenic RAS mutations, we have used small molecules to probe specific weaknesses of this cancerous network through synthetic lethal screening. One molecule identified in these screens, RSL3, attracted interest as a candidate for target identification studies because of its potent lethality and potentially unique mechanism of action. We used an affinity chromatography approach to directly isolate binding partners of RSL3 by modifying the molecules structure to incorporate various affinity tags. Through these experiments we ultimately identified a number of interesting candidate targets. Investigations validating these targets suggest that multi-targeted modulation of antioxidant and prostaglandin networks may be a mechanism for selectively killing cancers with oncogenic RAS. The identification of biological targets of small molecules poses a difficult challenge to the field of forward chemical genetics. Thus, we attempted to optimize a unique method for target identification, the yeast three-hybrid system (Y3H), which detects small molecule-protein interactions through a transcriptional assay in vivo. We created a version of our Y3H system that incorporated a covalent anchor and compared it with the existing state-of-the-art, which uses a high affinity non-covalent anchor. Transcriptional assays indicated our new system was functional, but surprisingly could not improve upon the original Y3H system. These results highlight the complexities of manipulating ligand-receptor interactions in vivo.

Unique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained– suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms. / text

Mobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency. / text

Homologous recombination (HR) is a faithful mechanism for the repair of double-stranded DNA breaks (DSBs) and plays a critical role in maintaining the integrity of genomic DNA. The product of the Breast Cancer Susceptibility 2 (BRCA2) gene functions as a recombination mediator in HR-directed repair of DSBs. BRCA2 interacts directly with RAD51, the central recombinase of HR, through highly conserved repetitive motifs of 30-40 amino acids, named BRC repeats, and regulates the formation of the RAD51-ssDNA nucleoprotein filament. There is significant variability in the number of BRC repeats among taxa. However, all mammalian BRCA2 orthologs have eight BRC repeats, which display different characteristics in in vitro studies of RAD51-ssDNA nucleoprotein filament. To test the importance of the number of BRC repeats and to evaluate the role of individual BRC repeats in HR, BRCA2 variants bearing different combinations of BRC repeats were generated using BAC-recombineering, expressed in murine hybridoma cells, and assayed for the ability to stimulate HR using a gene targeting assay. The BRCA2 variant bearing BRC repeats 1 to 4 decreased the efficiency of HR and increased the level of Rad51 protein, whereas the BRCA2 variant bearing BRC repeats 5 to 8 significantly stimulated HR, but had no effect on the level of Rad51. These results supported the hypothesis that BRC repeats are not functionally equivalent, but rather have different, perhaps reinforcing functions in HR. / Canadian Institutes of Health Research

This work was designed as a proof-of-principle concept or prototype to show the effect of protein-assisted targeting of DNA to specific genomic loci. Two strategies were employed to deliver the DNA with the aim that once inside the cell the DNA would be delivered to the target sequence by the assistance of a protein. In our case, the chosen protein was the site-specific meganuclease I-SceI. The first strategy described herein was to bind the targeting DNA to I-SceI by the use of a fusion protein between I-SceI and a known DNA-binding domain, the GAL4-DBD. The second strategy involved using a DNA aptamer to I-SceI to link the targeting DNA and I-SceI. Testing in vivo revealed that in our human cells (HEK-293) single-stranded DNA was more efficient at gene targeting than double-stranded DNA. In order for the first strategy to work, we needed to have some region of double-stranded DNA. We found that in human cells, it was better for gene targeting to have that double-stranded DNA on the 5’ side of our targeting DNA. We also used gel shift assays to confirm binding by our candidate DNA-binding domain, the GAL4-DBD. We were unable to detect expression of the fusion protein of I-SceI and the GAL4-DBD. For the second strategy we were able to construct an aptamer to I-SceI using a variant of the systematic evolution of ligands by exponential enrichment (SELEX). The I-SceI aptamer was synthesized as part of a longer DNA molecule containing homology to a target locus. Using this chimeric oligonucleotide (part aptamer, part DNA repair region) testing was done in both yeast and human cells. Aside from instances where the aptamer’s secondary structure may have been compromised, the aptamer containing oligonucleotide stimulated repair at a rate 2 to 15-fold higher than the non-selected control sequence. These experimental results show that by delivering targeting DNA within close proximity to the site of modification, gene targeting frequencies can be increased.

Thesis (M.S.)--University of North Texas, Dec., 2007. / Title from title page display. Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, December 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--University of Delaware, 2007. / Principal faculty advisor: Eric B. Kmiec, Dept. of Biological Sciences. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22707号 / 農博第2423号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5300(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM