Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

DNA replication demands extraordinary specificity and efficiency of catalysis from a DNA polymerase. Previous studies on several DNA polymerases suggested that a rate-limiting conformational change preceding chemistry accounts for the high specificity following the induced fit mechanism. However, the identity of this rate-limiting conformational change and how it contributes to the fidelity is still under debate. An important study of T7 DNA polymerase performed by Tsai and Johnson using a conformationally sensitive fluorophore (CSF) characterized a conformational change directly and presented a new paradigm for nucleotide selectivity. This thesis describes work to further characterize the underlying molecular basis regulating the conformational change by a combination of site-directed mutagenesis, transient kinetics and crystallography. One flexible segment (gly-ala-gly) within the fingers domain was mutated to (ala-alaala). The kinetic analysis on this mutant showed that the mutations decreased the forward rate of the conformational change reported by the fluorophore about 1200-fold but there was no significant change on the reverse rate. The data suggested that the movement of the fingers domain is not a rigid body motion but may be complex due to the movements of various helices within the fingers domain. Quantification of the kinetics of incorporation of correct and incorrect base pairs showed the decrease of fidelity mainly was from the decreased forward rate during correct nucleotide incorporation. The roles of three active site residues, K522, H506, and R518, which form polar interactions with [alpha]-,[beat]- and [gamma]-phosphates of the incoming nucleotide respectively, in conformational change and catalysis were also characterized. All the mutants showed a slower conformational change than the wild type enzyme. After this conformational change, there was a rate limiting step with a rate comparable to kpol measured by quench-flow experiments. Correct nucleotide binding caused an increase in fluorescence, suggesting that the conformational change of the fingers domain delivers incoming nucleotide to a misaligned status even for a correct nucleotide with each of the mutants. The data suggested that active site residues play important roles in maintaining a fast conformational change and an accurate alignment of the active site during correct nucleotide incorporation. Yellow crystals of CSF-labeled T7 DNA polymerase with DNA and correct nucleotide (closed complex), incorrect nucleotide (misaligned complex) or no nucleotide (open complex) were grown to good size and diffracted to 3 Å during X-ray data collection. The structures of these complexes are still under refinement. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [114]-131).

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.