Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled cysteine mutant of the 45 protein. This protein possessed unaltered function yet produced substantial changes in probe fluorescence intensity upon interacting with other components of the holoenzyme. These fluorescence changes provide insight into the role of ATP hydrolysis in holoenzyme assembly. Using either ATP or the non-hydrolyzable ATP analog, adenosine 5'-O-(3-thiophosphate), events in holoenzyme assembly were assigned as either dependent or independent of ATP hydrolysis. A holoenzyme assembly mechanism is proposed in which the 44/62 complex mediates the association of the 45 protein with DNA in an ATP-dependent manner not requiring ATP hydrolysis. Upon ATP hydrolysis, the 44/62 complex triggers a conformational change in the 45 protein that may be attributed to the clamp loading onto DNA.
Sexton
D J
DJ
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. sjb1@psu.edu
Carver
T E
TE
Berdis
A J
AJ
Benkovic
S J
SJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000235:genetics*; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D010069:Oxadiazoles / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D010069:Oxadiazoles; C102356:T7C-45 protein, Bacteriophage T4; C102357:T7C-45 protein-ANBD; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase