DOUBLE-STRAND BREAK REPAIR PATHWAYS IN DNA STRUCTURE-INDUCED GENETIC INSTABILITY

Genetic instability in mammalian cells can occur by many different mechanisms. In the absence of exogenous sources of DNA damage, the DNA structure itself has been implicated in genetic instability. When the canonical B-DNA helix is naturally altered to form a non-canonical DNA structure such as a Z-DNA or H-DNA, this can lead to genetic instability in the form of DNA double-strand breaks (DSBs) (1, 2). Our laboratory found that the stability of these non-B DNA structures was different in mammals versus Escherichia coli (E.coli) bacteria (1, 2). One explanation for the difference between these species may be a result of how DSBs are repaired within each species. Non-homologous end-joining (NHEJ) is primed to repair DSBs in mammalian cells, while bacteria that lack NHEJ (such as E.coli), utilize homologous recombination (HR) to repair DSBs. To investigate the role of the error-prone NHEJ repair pathway in DNA structure-induced genetic instability, E.coli cells were modified to express genes to allow for a functional NHEJ system under different HR backgrounds. The Mycobacterium tuberculosis NHEJ sufficient system is composed of Ku and Ligase D (LigD) (3). These inducible NHEJ components were expressed individually and together in E.coli cells, with or without functional HR (RecA/RecB), and the Z-DNA and H-DNA-induced mutations were characterized. The Z-DNA structure gave rise to higher mutation frequencies compared to the controls, regardless of the DSB repair pathway(s) available; however, the type of mutants produced after repair was greatly dictated on the available DSB repair system, indicated by the shift from 2% large-scale deletions in the total mutant population to 24% large-scale deletions when NHEJ was present (4). This suggests that NHEJ has a role in the large deletions induced by Z-DNA-forming sequences. H-DNA structure, however, did not exhibit an increase in mutagenesis in the newly engineered E.coli environment, suggesting the involvement of other factors in regulating H-DNA formation/stability in bacterial cells. Accurate repair by established DNA DSB repair pathways is essential to maintain the stability of eukaryotic and prokaryotic genomes and our results suggest that an error-prone NHEJ pathway was involved in non-B DNA structure-induced mutagenesis in both prokaryotes and eukaryotes.

Subnanometer-resolution structures of the grass carp reovirus core and virion.

Grass carp reovirus (GCRV) is a member of the Aquareovirus genus of the family Reoviridae, a large family of double-stranded RNA (dsRNA) viruses infecting plants, insects, fishes and mammals. We report the first subnanometer-resolution three-dimensional structures of both GCRV core and virion by cryoelectron microscopy. These structures have allowed the delineation of interactions among the over 1000 molecules in this enormous macromolecular machine and a detailed comparison with other dsRNA viruses at the secondary-structure level. The GCRV core structure shows that the inner proteins have strong structural similarities with those of orthoreoviruses even at the level of secondary-structure elements, indicating that the structures involved in viral dsRNA interaction and transcription are highly conserved. In contrast, the level of similarity in structures decreases in the proteins situated in the outer layers of the virion. The proteins involved in host recognition and attachment exhibit the least similarities to other members of Reoviridae. Furthermore, in GCRV, the RNA-translocating turrets are in an open state and lack a counterpart for the sigma1 protein situated on top of the close turrets observed in mammalian orthoreovirus. Interestingly, the distribution and the organization of GCRV core proteins resemble those of the cytoplasmic polyhedrosis virus, a cypovirus and the structurally simplest member of the Reoviridae family. Our results suggest that GCRV occupies a unique structure niche between the simpler cypoviruses and the considerably more complex mammalian orthoreovirus, thus providing an important model for understanding the structural and functional conservation and diversity of this enormous family of dsRNA viruses.

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^

Policy analysis of the Americans with Disabilities Act of 1990 and federal funding concerning barrier-free playgrounds

The Americans with Disabilities Act (ADA) of 1990 was created to prohibit discrimination against disabled persons in our society. The goal of the ADA as a comprehensive civil rights law is to "ensure equal opportunity and complete participation, independent living and economic self-sufficiency" for disabled persons (U.S. Department of Justice, 2008). As part of Title II and III of the ADA, states and local governments are required to provide people with disabilities the same chance to engage in and benefit from all programs and services including recreational facilities and activities as every other citizen. Recreational facilities and related structures must comply with accessibility standards when creating new structures or renovating existing ones. Through a systematic literature review of articles accessed through online databases, articles relating to children with disabilities, their quality of life and their experience gained through play were reviewed, analyzed and synthesized. Additionally, the ADA's Final Rule regarding accessible playgrounds was evaluated through a descriptive analysis which yielded the following five components relating the importance of barrier-free playgrounds to children with disabilities: appropriate dimensions for children, integration of the play area, variety of activity and stimulation, availability of accessible play structures to communities, and financial feasibility. These components were used as evaluation criteria to investigate the degree to which the ADA's Final Rule document met these criteria. An evaluation of two federal funding sources, the Urban Parks and Recreation Renewal Program (UPARR) and the Land and Water Conservation Fund (LWCF), was also conducted which revealed three components relating the two programs' ability to support the realization of the ADA's Final Rule which included: current budget for the program, ability of local communities to attain funds, and level of ADA compliance required to receive funding. Majority of the evaluation of the Final Rule concluded it be adequate in development of barrier-free playgrounds although there are some portions of the guidelines that would benefit from further elucidation. Both funding programs were concluded to not adequately support the development of barrier-free playgrounds and therefore it was recommended that their funding be re-instated or increased as necessary. ^