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.

Abstract:

A multisubunit ring-shaped protein complex is used to tether the ...
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.

Polymerases:

T4

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