Yes / Non-healing wounds are a major area of unmet clinical need remaining problematic to treat. Improved understanding of pro-healing mechanisms is invaluable. The enzyme arginase1 is involved in pro-healing responses with its role in macrophages best characterized. Arginase1 is also expressed by keratinocytes; however, arginase1 function in these critical wound repair cells is not understood. We characterized arginase1 expression in keratinocytes during normal cutaneous repair and reveal de novo temporal and spatial expression at the epidermal wound edge. Interestingly, epidermal arginase1 expression was decreased in both human and murine delayed healing wounds. We therefore generated a keratinocyte specific arginase1-null mouse model (K14-cre;Arg1fl/fl) to explore arginase function. Wound repair, linked to changes in keratinocyte proliferation, migration and differentiation, was significantly delayed in K14-cre;Arg1fl/fl mice. Similarly, using the arginase inhibitor nor-NOHA, human in vitro and ex vivo models further confirmed this finding, revealing the importance of the downstream polyamine pathway in repair. Indeed, restoring the balance in arginase1 activity via addition of putrescine, proved beneficial in wound closure. In summary, we demonstrate that epidermal arginase1 plays a, to our knowledge, previously unreported intrinsic role in cutaneous healing, highlighting epidermal arginase1 and downstream mediators as potential targets for the therapeutic modulation of wound repair.

DNA repair is not a single mechanism found within cells. There exists numerous different DNA repair mechanisms that function within every type of cell. The majority of these mechanisms risk accumulating mutations. However, there are a few repair mechanisms that are known to be error-free and one of these is direct reversal repair. This study focused on two proteins highly involved in direct reversal DNA repair--ALKBH2 and ALKBH3. Previous studies have shown that in mice, these two proteins play a significant role in preventing and repairing DNA damage due to methylation as well as decreasing the frequency of mutagenic alkyl adducts. The goal of this study was to characterize the roles of the direct reversal repair proteins in human cells. We expected to see a similar phenotype to that of the Alkbh2 and Alkbh3-deficient mice. Telomerase immortalized human skin fibroblasts were targeted for the ALKBH2 and ALKBH3 alleles using a RNA-guided CRISPR-Cas9 construct that was designed to induce double stranded DNA breaks within the exons and disrupt the open reading frame, eliminating protein activity. Isolated clones were analyzed using fragment analysis and DNA sequencing to characterize any alterations in the open reading frame of the genes. Through sequencing analysis, results showed that one clone was successfully targeted for one of the ALKBH3 alleles with a single nucleotide insertion in its sequence, causing a disruption of the open reading frame. Though the ultimate goal of the experiment was not attained, we concluded that HTERTG fibroblasts can be expanded to serve as a model in which to construct targeted human cell lines that have near normal karyotypes.

No description available.

published_or_final_version / Applied Statistics / Master / Master of Social Sciences

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.

No description available.

No description available.