Opening of a monomer-monomer interface of the trimeric bacteriophage T4-coded GP45 sliding clamp is required for clamp loading onto DNA.
The replication system of bacteriophage T4 uses a trimeric ring-shaped processivity clamp (gp45) to tether the replication polymerase (gp43) to the template-primer DNA. This ring is placed onto the DNA by an ATPase-driven clamp-loading complex (gp44/62) where it then transfers, in closed form, to the polymerase. It generally has been assumed that one of the functions of the loading machinery is to open the clamp to place it around the DNA. However, the mechanism by which this occurs has not been fully defined. In this study we design and characterize a double-mutant gp45 protein that contains pairs of cysteine residues located at each monomer-monomer interface of the trimeric clamp. This mutant protein is functionally equivalent to wild-type gp45. However, when all three monomer-monomer interfaces are tethered by covalent crosslinks formed (reversibly or irreversibly) between the cysteine pairs these closed clamps can no longer be loaded onto the DNA nor onto the polymerase, effectively eliminating processive strand-displacement DNA synthesis. Analysis of the individual steps of the clamp-loading process shows that the ATPase-dependent interactions between the clamp and the clamp loader that precede DNA binding are hyperstimulated by the covalently crosslinked ring, suggesting that binding of the closed ring induces a futile, ATP-driven, ring-opening cycle. These findings and others permit further characterization and ordering of the steps involved in the T4 clamp-loading process.
Latham
G J
GJ
Institute of Molecular Biology, Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
Dong
F
F
Pietroni
P
P
Dozono
J M
JM
Bacheller
D J
DJ
von Hippel
P H
PH
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017122:Bacteriophage T4 / Q000378:metabolism*; D001704:Biopolymers; D003432:Cross-Linking Reagents / Q000737:chemistry; D004279:DNA, Viral / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4; D000251:Adenosine Triphosphatases