Building a replisome solution structure by elucidation of protein-protein interactions in the bacteriophage T4 DNA polymerise holoenzyme

Assembly of DNA replication systems requires the coordinated actions of man y proteins. The multiprotein complexes formed as intermediates on the pathw ay to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual protei ns. By using a variety of solution-phase techniques, we have elucidated add itional information about the solution structure of the bacteriophage T4 ho loenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fl uorescence resonance energy transfer, analytical ultracentrifugation, and i sothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoe nzyme assembly, but only one of these binding modes is found in the final h oloenzyme. Present and previous solution interaction data were used to buil d a model of the holoenzyme that is consistent with these data.