Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. I. Conformational changes within the gp44/62-gp45-ATP complex during clamp loading.
A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems. This "sliding clamp" interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process. Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45). Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process. Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle. A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process.
Pietroni
P
P
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, USA.
Young
M C
MC
Latham
G J
GJ
von Hippel
P H
PH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005464:Fluorobenzenes / Q000494:pharmacology; D046911:Macromolecular Substances; D008301:Maleimides / Q000494:pharmacology; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011487:Protein Conformation; D013604:T-Phages / Q000201:enzymology*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D005464:Fluorobenzenes; D046911:Macromolecular Substances; D008301:Maleimides; C102356:T7C-45 protein, Bacteriophage T4; D015534:Trans-Activators; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; C073711:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase