Beschreibung:
<jats:p>Sliding clamps are found in all domains of life and are essential for efficient DNA replication, which is required every time a cell divides, as well as for coordinating traffic on DNA. The <jats:italic>Escherichia coli</jats:italic> (<jats:italic>E. coli</jats:italic>) beta‐clamp is a ring‐shaped protein consisting of two identical monomers encoded by the <jats:italic>dnaN</jats:italic> gene that associates with DNA polymerase III and facilitates the high degree of processivity required for DNA replication. Sliding clamp proteins open and are loaded onto specific DNA structures by clamp loaders in the presence of ATP. We aim to gain insights into the contributions of different domains to the function of beta using a linked‐beta‐clamp that constrains the homodimer at one of two interfaces. Usually the homo‐dimeric structure of the beta‐clamp allows it to act as a molecular tool belt, binding more than one protein simultaneously at specific protein interaction sites on the clamp. Thus, a better understanding of the sliding clamps' role in the process of DNA damage tolerance will be gathered by investigating whether one interface or binding site is adequate for DNA loading or for other protein interactions. To create this construct, the linker length and sequence, as well as expression conditions, were optimized. The linked‐beta proteins were characterized through a thermal denaturation assay, and showed comparable thermal stability to wild‐type beta. In an ATPase assay, similar amounts of inorganic phosphate were detected in reactions containing either linked‐beta or wild‐type beta, indicating that the clamp loader can interact with both versions of the beta clamp. Currently, we are investigating whether linked and wild‐type beta improve the processivity of a polymerase in a primer extension assay. We are now constructing and characterizing variants of wild‐type beta and linked‐beta to test the importance of residues at only one of the interfaces or partner protein binding sites.</jats:p>