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 (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 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 topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at 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. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.
Trakselis
M A
MA
Department of Chemistry, 414 Wartik Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.
Alley
S C
SC
Abel-Santos
E
E
Benkovic
S J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005453:Fluorescence; D020035:Holoenzymes / Q000737:chemistry*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D012996:Solutions; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
D020035:Holoenzymes; D012996:Solutions; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase