Abstract:

The Escherichia coli ? processivity clamp interacts with multiple factors involved in DNA replication, repair and damage tolerance. This suggests a role for the clamp in the coordination of these processes. A conserved hydrophobic cleft on the surface of ? is thought to interact with all other clamp-binding factors. The ? clamp exists as a homodimer and each dimer contains two hydrophobic clefts. This work focuses on the role of these hydrophobic clefts in interactions with partner proteins. Biochemical assays were conducted to characterize ? clamp interactions with DNA polymerases and the clamp loader complex.

Deletion of the C-terminal five residues of ? (?C ) severely impaired interactions of the clamp with the clamp loader complex and more specifically the d subunit. In order to determine whether both clefts were required for loading clamp onto DNA and removal of clamp from DNA after replication was complete, a method was developed for purification of heterodimeric clamp protein comprised of one wild type subunit and one ?C subunit (? + /?C ). The ?+ /? C heterodimer interacted normally with the clamp loader complex, and was loaded onto DNA slightly more efficiently than was wild type ?. However, ? + /?C was severely impaired for unloading from DNA using either clamp loader or the o subunit alone. Taken together, these finding indicate that a single cleft in the ? clamp is sufficient for loading but that both clefts are required for unloading clamp from DNA after replication is completed.

Each of the five DNA polymerases in E. coli has also been reported to interact with the ? clamp and are predicted to bind to the conserved hydrophobic cleft on ?. In addition to the cleft, other surfaces on the clamp are also involved in these interactions. I have examined interactions of four of the five DNA polymerases, DNA polymerases I, II III and IV, with the ? clamp. DNA polymerase V was not surveyed because the reagent was unavailable. Also, a complex between DNA polymerase I and ? was not detected under the conditions of these assays. DNA polymerases II, III and IV were further examined with a collection of ? clamp mutants on the surface of the clamp that contacts all of the partner proteins. These mutations were in both the hydrophobic cleft and noncleft sites to map regions that each polymerase interacts with the clamp.

As predicted, the hydrophobic cleft was an important shared interaction site for all three of the polymerases tested. I have identified a rule for the orientation of DNA polymerases on processivity clamps that was predicted by how the polymerase binds to the cleft. This was based on the placement of the clamp binding motif (CBM) on the polymerase and the path that DNA follows as it exists the polymerase through the center of the clamp. In summary, each of the DNA polymerases appears to interact with the clamp in a unique way, despite the common binding site at the hydrophobic cleft. This could be related to function and could also be a basis for differential recruitment of the polymerases to the DNA through the ? clamp.