Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp.
The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles DNA and tethers the replicase to the template. The beta dimer is assembled around DNA by the gamma complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and beta is identified as mediated through the delta subunit of the gamma complex. The delta subunit appears to contact the face of the beta dimer ring that contains the two C termini. Surprisingly, ATP is required for the gamma complex to bind beta, but not for delta to bind beta. This indicates that delta is buried in the gamma complex and suggests a role for ATP in exposing delta for interaction with beta. A protease protection assay has been developed to specifically probe the delta subunit within the gamma complex. The results of the assay are consistent with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it. The implication of these key features to the clamp loading mechanism of the gamma complex is discussed.
Naktinis
V
V
Microbiology Department, Cornell University Medical College, New York, New York 10021, USA.
Onrust
R
R
Fang
L
L
O'Donnell
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D002876:Chromosomes, Bacterial; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D011487:Protein Conformation
D004269:DNA, Bacterial; D007527:Isoenzymes; D008670:Metals; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D004258:DNA Polymerase III