DNA replication and repair are vital processes for all domains of life as they are the basis of biological inheritance. DNA polymerase processivity factors, including sliding clamps, play an important role in both DNA damage tolerance and repair. The Escherichia coli beta-clamp is a ring-shaped protein homodimer encoded by the dnaN gene that associates with DNA polymerase III and facilitates the high degree of processivity needed for efficient DNA replication. Sliding clamp proteins open and load 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. To create this construct, different linker lengths and sequences, as well as different expression conditions, were investigated in various E. coli strains. The linked-beta proteins were purified and characterized relative to wild-type beta through a thermal denaturation assay, and showed comparable thermal stability. 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 version 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. With this research, we aim to provide a deeper understanding of the role of the sliding clamp in DNA replication, damage tolerance, and repair.

Dynamics of the E. coli beta clamp and its influence on DNA loading

253rd National Meeting (2017)

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