Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme .1. 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 crosslinker 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.