Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme.
The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.
Rockefeller University and Howard Hughes Medical Institute, Laboratory of DNA Replication, New York, New York 10021, USA.
J
J
Stewart
M M
MM
Hingorani
Z
Z
Kelman
M
M
O'Donnell
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D013997:Time Factors
D009532:Nickel; D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III