Janina Cramer

The Coiled Coil Region (Amino Acids 129–250) of the Tumor Suppressor Protein Adenomatous Polyposis Coli (APC) ITS STRUCTURE AND ITS INTERACTION WITH CHROMOSOME MAINTENANCE REGION 1 (Crm-1)

The APC (adenomatous polyposis coli) tumor suppressor protein has many different intracellular functions including a nuclear export activity. Only little is known about the molecular architecture of the 2843-amino acid APC protein. Guided by secondary structure predictions we identified a fragment close to the N-terminal end, termed APC-(129–250), as a soluble and protease-resistant domain. We solved the crystal structure of APC-(129–250), which is monomeric and consists of three α-helices forming two separate antiparallel coiled coils. APC-(129–250) includes the nuclear export signal NES-(165–174) at the C-terminal end of the first helix. Surprisingly, the conserved hydrophobic amino acids of NES-(165–174) are buried in one of the coiled coils and are thus not accessible for interaction with other proteins. We demonstrate the direct interaction of APC-(129–250) with the nuclear export factor chromosome maintenance region 1 (Crm-1). This interaction is enhanced by the small GTPase Ran in its activated GTP-bound form and also by a double mutation in APC-(129–250), which deletes two amino acids forming two of the major interhelical interactions within the coiled coil. These observations hint to a regulatory mechanism of the APC nuclear export activity by NES masking.

Pre-steady-state kinetic characterization of the DinB homologue DNA polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is ∼10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s-1 for dTTP, dATP, dGTP, and dCTP (at 25 °C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (Kd ∼600 μm), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an “induced fit” mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.