Brun, Jan2013-11-082013-11-0820072007Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7315.http://hdl.handle.net/10393/29466http://dx.doi.org/10.20381/ruor-12970Ubiquitination is a highly regulated post-translational modification that impinges on a variety of cellular processes. Commonly known for its role in proteolysis, recent studies emphasize the versatility of ubiquitin in non-proteolysic aspects of cell function. Some of these proteolysic-independent roles have been highlighted in recent publications as being critical mediators in stress response pathways including those of DNA repair and NF-kappaB activation. In these seemingly unrelated pathways it has been shown that a noncanonical linear chain linked through lysine 63(K63) is required in the assembly of multi-subunit complexes which allow eukaryotes to tolerate DNA damage or elicit an immune response. Because the functionality of ubiquitin chains is constrained by length and topology, alteration of these properties greatly alters these cellular pathways. In order to understand the role of these chains in mammalian cells we introduced mutant ubiquitin proteins that interfere with specific properties of ubiquitin chain assembly. These dominant negative mutants revealed interesting biological effects. Here we demonstrate that in human cells, as in yeast, PCNA (proliferating cell nuclear antigen) is polyubiquitinated via K63. Disruption of K63-linked chains impairs the error-free arm of a DNA damage tolerance (DDT) pathway resulting in an increased reliance on error-prone translesion synthesis (TLS), which has significant implications on mutagenesis particularly and in response to common environmental carcinogens such as UV light and BPDE. As a result of our cell culture studies, we have generated transgenic mice expressing the K63R mutant form of ubiquitin to more directly test its roles in cancer development and impaired NF-kappaB induction in vivo. Recently, several members of error-free DDT including POLeta, SHPRH, hRAD6, hRAD18 have been linked to cancer and the likelihood of chemotherapeutic success in patients. Accordingly, future drug targeting of the error-free DDT pathway in cancer could be of tremendous therapeutic benefit.307 p.enChemistry, Biochemistry.Thesis