Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (g p45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polym erase holoenzyme is a multistep process. A partially opened toroid-shaped g p45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologica lly link gp43 to DNA, effectively increasing the processivity of DNA replic ation. Stopped-flow fluorescence resonance energy transfer was used to inve stigate the opening and closing of the gp45 ring during holoenzyme assembly . By using two site-specific mutants of gp45 along with a previously charac terized gp45 mutant, we tracked changes in distances across the gp45 subuni t interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring a t one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Ad dition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformat ion. The final holoenzyme is formed by the addition of gp43, which causes g p45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to i nteract with the C-terminal tail of gp43, providing a point of contact betw een gp45 and gp43. This study further defines the dynamic process of bacter iophage T4 polymerase holoenzyme assembly.