Dual role of the 44/62 protein as a matchmaker protein and DNA polymerase chaperone during assembly of the bacteriophage T4 holoenzyme complex.
Processive DNA synthesis in the bacteriophage T4 system requires the formation of a holoenzyme complex composed of the T4 DNA polymerase and the 44/62 and 45 accessory proteins. While ATP hydrolysis by the 44/62 protein is essential for holoenzyme formation, the role of the sliding clamp or processivity factor is attributed to the 45 protein. Beyond the need for ATP hydrolysis, the exact role of the 44/62 protein in complex assembly has not been clearly defined. In this paper, we have investigated the kinetics of complex assembly in the presence of both saturating and substoichiometric concentrations of the 44/62 protein. Under saturating conditions, complex assembly is 100% efficient, with all of the polymerase bound in a processive complex. Under conditions of limiting 44/62 protein, the 44/62 protein can act catalytically to assemble the 45 protein and polymerase into a productive complex. However, kinetic simulations indicate that a significant fraction of polymerase is sequestered in a nonproductive complex with the 45 protein. Thus, a second role for the 44/62 protein during complex assembly is that of a chaperone protein to ensure productive pol.45.DNA complex formation. We have also investigated the stability of the 45 protein on the DNA. The off rate of 0.003 s-1 for the 45 protein closely parallels that of the holoenzyme complex. Therefore, disassembly of the complex appears to involve the coordinated dissociation of both the 45 protein and the polymerase from the DNA.
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
B F
BF
Kaboord
S J
SJ
Benkovic
D017122:Bacteriophage T4 / Q000737:chemistry*; D001483:Base Sequence; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D007700:Kinetics; D018832:Molecular Chaperones / Q000502:physiology*; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000378:metabolism* / Q000502:physiology*
D018832:Molecular Chaperones; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase