<p>Translesion synthesis (TLS) is a specialized type of DNA repair for bypassing DNA damage at the stalled replication fork. Because the TLS polymerases (mainly from the Y-family of polymerases) are characterized by more open active sites in order to accommodate the lesions, they are inherently more mutagenic than the replicative polymerases. Although essential for cell survival and in tolerating DNA damage, the access of the TLS polymerases to the replication fork must be tightly controlled. This regulation occurs in part through the ubiquitination state of the processivity factor PCNA. Damage-induced monoubiquitination of PCNA serves in part as the regulatory switch between replicative and translesion polymerases. Highly conserved ubiquitin-binding domains, the ubiquitin-binding zinc finger (UBZ) domain and the ubiquitin-binding motif (UBM), within the C-termini of the Y-family polymerases provide for an increased affinity of the polymerases to PCNA after damage to promote TLS. In order to determine the molecular basis for ubiquitin recognition by the TLS polymerases, we solved the solution structures of the human pol &#951; UBZ domain, the human pol &#953; UBM2 domain, and the human pol &#953; UBM2-ubiquitin complex. </p><p>The UBZ domain adopts a classical C2H2 zinc finger structure characterized by a &#946;&#946;&#945; fold, similar to the DNA-binding zinc finger proteins. Nuclear magnetic resonance titration mapped the binding interfaces between UBZ and ubiquitin to the &#945;-helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single &#945;-helix, in a manner similar to the inverted ubiquitin-interacting motif, its structure is distinct from previously characterized ubiquitin-binding domains. The pol &#951; UBZ domain represents a novel member of the C2H2 zinc finger family that interacts with ubiquitin to regulate translesion synthesis. </p><p>In contrast to the human UBZ domain, the yeast UBZ domain lacks one of the conserved cysteines necessary for zinc coordination, leading many to propose that it is a "zincless" zinc finger. We used biophysical methods to characterize the UBZ domains from human and yeast pol &#951; and to highlight differences between their structures and modes of ubiquitin binding. Like the human UBZ domain, the yeast UBZ domain binds zinc, which contributes to its secondary structure formation. In contrast to the human UBZ domain, the yeast UBZ domain binds to ubiquitin in a zinc-independent manner. Correspondingly, mutations in the zinc-coordinating residues of the yeast UBZ domain do not impair the polymerase's response to DNA damage.</p><p>We also investigated the structural elements and mechanism of ubiquitin recognition of the ubiquitin-binding motif (UBM) found in pol &#953; and Rev1. The solution structures of the C-terminal UBM of human pol &#953; and its complex with ubiquitin were solved. The UBM is a novel ubiquitin-binding domain that binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A of ubiquitin abolishes UBM binding. Human pol &#953; contains two functional UBMs, both of which contribute to replication foci formation. In contrast, only the second UBM of <italic>Saccharomyces cerevisiae</italic> Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations impairing the UBM-ubiquitin interaction also disrupt foci formation of pol &#953; and the DNA damage response of Rev1 <italic>in vivo</italic>, showing the significant role for the UBM in regulating TLS.</p><p>The structures of the UBZ domain and the UBM and their recognition of ubiquitin are different and distinct from other ubiquitin-binding domains. Their highly specific and unique associations with ubiquitin are critical for TLS regulation and further add to the diverse base of ubiquitin-binding domains and their role in mediating cellular functions.</p> / Dissertation

Therapeutic antibodies have achieved exceptional clinical success in the treatment of cancer and other human diseases. Now, new approaches are required to enhance the potency of antibodies to further increase the number of patients responding to therapy. By engineering the antibody Fc domain through mutation of the amino acid sequence, binding affinity to activating or inhibitory Fc receptors on effector cells can be increased to modulate the cellular immune response. However, attaining selectivity for closely related Fc receptors has proved challenging and the technique has not been applied to access the function of antibody isotypes other than IgG. Here we present new methods for enhancing antibody potency using both hybrid IgA/G and aglycosylated Fc domains. In the first instance, a chimeric antibody Fc domain has been created by combining residues from IgA with those from IgG. The new variant, MutD, introduces binding to FcαRI while retaining affinity for certain members of the FcγR family. ADCC assays show MutD, when part of a full length trastuzumab antibody against Her2 antigen, can kill Her2-overexpressing tumor cell lines as effectively as IgA antibodies. Moreover, MutD shows improved assembly compared to IgA and thus provides access to potent FcαRI function while overcoming the expression and purification barriers that have limited the use of IgA as a therapeutic. Alternatively, aglycosylated antibodies may be engineered for exceptional effector function. Glycans anchored to residue N297 of the antibody IgG Fc domain are typically critical in mediating binding toward the FcγRs. Yet, using a full length bacterial IgG display system, we have isolated aglycosylated Fc1004 with mutations that confer a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous inhibitory receptor, FcγRIIb. Incorporation of this engineered Fc into trastuzumab resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with medium Her2-expressing cancer cells. In vivo testing of Fc1004 using NOD/SCID mouse model, reconstituted by adoptive transfer of leukocytes from FcγRIIa-R131 homozygous donors, showed a promising reduction in tumor burden in SkBr-3 Her2+ xenografts. / text

No description available.

The basic unit of life is the cell, from single-cell bacteria to the largest creatures on the planet. All cells have DNA, which contains the blueprint for proteins. This information is transported in the form of messenger RNA from the genome to ribosomes where proteins are produced. Proteins are the main functional constituents of the cell, they usually have one or several functions and are the main actors in almost all essential biological processes. Proteins are what make the cell alive. Proteins are found as solitary units or as part of large complexes. Proteins can be found in all parts of the cell, the most common place being the cytoplasm, a central space in all cells. They are also commonly found integrated into or attached to various membranes. Membranes define the cell architecture. Proteins integrated into the membrane have a wide number of responsibilities: they are the gatekeepers of the cell, they secrete cellular waste products, and many of them are receptors and enzymes. The main focus of this thesis is the study of protein interactions, from the molecular level up to the protein domain level. In paper I use reoccurring local protein structures to try and predict what sections of a protein interacts with another part using only sequence information. In papers II and III we use a randomization approach on a membrane protein motif that we know interacts with a sphingomyelin lipid to find other candidate proteins that interact with sphingolipids. These are then experimentally verified as sphingolipid-binding. In the last paper, paper IV, we look at how protein domain interaction networks overlap and can be evaluated. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Typescript (Photocopy) / Bibliography: leaf 75 / 75 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pure Mathematics, 1985?

Dissertation (Ph.D.)--Auburn University, 2007. / Abstract. Includes bibliographic references (p.58-59).

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references (p.25).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 96-111).

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains v, 88 p. Includes bibliographical references (p. 87-88). Available online via OhioLINK's ETD Center

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2009. / Committee Chair: David Anderson; Committee Co-Chair: Alexander Gray; Committee Member: Anthony Yezzi; Committee Member: Hongyuan Zha; Committee Member: Justin Romberg; Committee Member: Ronald Schafer.