Mdm2 and MdmX are RING domain proteins that bind to and inhibit p53 trans-activation functions. Moreover, Mdm2 interacts with p53 and targets it for degradation. However, Mdm2 and MdmX function beyond a simple inhibition of p53, and increasing evidence suggests functions in regulation of target gene specificity by p53 as well as influencing gene expression through other transcription factors. In this dissertation we present two studies into the regulation of p53 target genes by MdmX and Mdm2. We found that MdmX is required for the full activation of the Mdm2 gene following cellular stress, but not of other p53 targets, such as p21. The resulting deficiency in Mdm2 induction after MdmX ablation results in impaired negative feedback loop, leading to prolonged p53 half life following DNA damage. In vitro, MdmX does not stimulate p53 interaction with Mdm2 promoter DNA. MdmX does, however, inhibit the binding of p53 to DNA to a much lesser extent than Mdm2 does. Strikingly, MdmX is required for optimal p53 binding to the Mdm2 promoter in vivo. Thus, we have described a new mechanism by which MdmX can suppress p53, which is through transcriptional activation of p53's principal negative regulator, Mdm2. PCNA is a DNA sliding clamp that is required for DNA replication and coordinates multiple aspects of DNA biology. It is reported to be both a direct activation target of p53, as well as an indirect repression target. We have examined the roles of Mdm2 and MdmX in the regulation of the PCNA gene.

The yeast repair DNA polymerase POL4 is part of the X family of DNA polymerases that function in DNA repair, specifically in gap filling for non-homologous end joining (NHEJ). POL4 is the only Pol X family polymerase in Saccharomyces cerevisiae and has been shown to have a role in creating deletions or insertions after double-strand break (DSB) repair by NHEJ, an error-prone mechanism. I investigated the role of the yeast POL4 genotype on mutations induced by a previously described yeast CRISPR/Cas9 system. In this system a galactose-inducible Cas9 is used in combination with a gRNA targeted to the yeast CAN1 gene. CAN1 mutations are easily selected by resistance to canavanine. Our initial analysis suggested that the pol4 mutant genotype resulted in larger deletions compared to wild-type POL4. However, we found that the Cas9 plasmid induced mutations in the absence of galactose indicating that basal Cas9 expression was sufficient for inducing mutations. This suggested that the mutations I identified were in many cases clonal and not independent from each other. This hypothesis was supported by similar mutations being enriched for within an experiment performed on the same day. After controlling for independence, we did not detect a POL4 genotype-dependent effect on mutation type. We also asked whether the POL4 genotype affected the overall mutation frequency in both haploid and diploid yeast. We found pol4 haploids had less overall CRISPR/Cas9 induced mutations than POL4 haploids while in diploids the overall mutation frequency was lower than in haploids but did not vary by genotype. Our results suggest that a requirement for POL4 in repair of DSBs caused by CRISPR/Cas9 is minor at best.

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Teleost immunoglobulin M (IgM), an 800 kDa tetramer, possesses considerable structural diversity due to the non-uniform disulfide polymerization of its halfmeric or monomeric subunits. However, to date, no plausible functional role for this diversity has been demonstrated or proposed. This research was, therefore, designed to investigate the possible functional role(s) for this diversity using the trout model. The possible relationship between this structural diversity and affinity was specifically addressed. The relationship between high levels of disulfide polymerization and high affinity was demonstrated by selective immunoadsorption and analysis of antibodies isolated during the process of affinity maturation. A pivotal determinative role of antigen/BCR affinity in conferring graded levels of disulfide bonding was demonstrated by the induction of high and mixed affinity antibodies from a single lymphocyte source in vitro. Additionally, transfer of immunopurified antibodies and labeled non-immune immunoglobulins revealed a direct effect of polymerization on antibody half-life, with selective removal of less polymerized Igs and/or retention of more fully polymerized Igs. Thus, this differential effect on half-life also results in an increase of average affinity, accentuating the process of affinity maturation. The converse, modulation of affinity by disulfide variation; however, could not be demonstrated.

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Primary disease-causing mutations have been identified in many inherited eye diseases. However, there is a wide range of variation in the penetrance and phenotypic expression of these mutations, making identification of the mutation alone insufficient in providing accurate prognosis and treatment. We studied two genetic eye diseases in which the primary disease-causing mutations have been identified: Leber hereditary optic neuropathy (LHON), a maternally transmitted disorder caused by mitochondrial mutations, and autosomal dominant retinal dystrophies (adRD), caused by a splice site mutation (IVS2+3A→T) in the peripherin/RDS gene. Incomplete penetrance in LHON and phenotypic variation in adRD suggest a role for modifier genes, identification of which would contribute towards the understanding of the pathophysiology of these diseases. We show evidence suggestive of linkage to the X-chromosome at two regions (Xq21.1 and Xq26-28) by chromosomal linkage analysis in an LHON family. Screening of coding regions in X-linked candidate genes, MAOB, TIMM8A, CDR1, LDOC1 and NDUFA excluded them as potential modifiers. We established founder effect for the RDS splice site mutation in nineteen putatively unrelated families with adRD by intragenic and STRP haplotype analysis. We identified an aberrant transcript among affected individuals harboring RDS splice site mutation by RT-PCR. We demonstrated three clinical phenotypes resulting from this mutation: pattern dystrophies, central areolar choroidal dystrophy and retinitis pigmentosa. We screened the coding region, promoter, and 3'UTR of RDS, and the coding regions of ROM1, rdCVF and GCAP1 as potential modifiers. Additional variants in RDS gene and ROM1 were excluded as modifiers. Sequence variations in GCAP1 and rdCVF were identified as potential modifiers in a few individuals harboring the splice site mutation. Our results suggest that the phenotypic manifestation of both mitochondrial and RDS splice site mutation is complex and is due to Oligogenic effects. Further studies with additional family members and functional studies are required to establish the linkage to X-chromosome in LHON and to determine the significance of GCAP1 and RdCVF sequence variants that were identified in adRD. In summary, a cumulative insight from the study of environmental, stochastic and genetic factors is required for the ultimate understanding and cure of these diseases.

Atomic structure determination of membrane proteins is an important problem. Because of the difficulties in crystallization and traditional NMR techniques using membrane proteins, other experimental methods are being developed and investigated. One such method, the solid-state NMR PISEMA (Polar Inversion Spin-Exchange at the Magic Angle) experiment, determines orientational contraints for the target membrane protein. These constraints can be used to build a high resolution atomic model. This dissertation presents a detailed analysis of the PISEMA experimental data set and how it can be used to derive atomic structure. One of the aspects of the data set is that it is degenerate, that is, the orientational information measured by the data does not uniquely describe atomic locations. Thus, there are many possible structures that would provide the same data. An important goal of this work is to enumerate and characterize these degeneracies throughout each phase of model building and provide computational tools to manage them. The process of building atomic models from PISEMA data involves three major steps: assignment, initial model building and atomic refinement. For each step we present new software that is intended to aid in the model building process.The tools are designed to be used consecutively with limited, though significant, human intervention. The last section of the dissertation presents an application of our tools to a new PISEMA data set, from which we derive the first high-resolution atomic structure of the transmembrane portion of the M2 proton channel from the Influenza A virus in the presence of amantadine.For this case, we explain both the data processing and decision making that determined the final atomic model. It is likely that this semi-automated procedure will be applicable to other transmembrane protein PISEMA data sets. / A Dissertation submitted to the Institute of Molecular BioPhysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Summer Semester, 2006. / Date of Defense: May 1, 2006. / Solid-State NMR, Structural Biology, Membrane Proteins, Influenza A / Includes bibliographical references. / Richard Bertram, Professor Directing Dissertation; Piyush Kumar, Outside Committee Member; Micheal S. Chapman, Committee Member; Timothy A. Cross, Committee Member; Jack R. Quine, Committee Member.

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