Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase.
The seven-protein bacteriophage T4 DNA replication complex can be manipulated in vitro to study mechanistic aspects of the elongation phase of DNA replication. Under physiological conditions, the processivity of DNA synthesis catalyzed by the T4 polymerase (gp43) is greatly increased by the interaction of this enzyme with its accessory proteins (gp44/62 and gp45) and the T4 single-stranded DNA binding protein (gp32). The assembly of this T4 holoenzyme requires hydrolysis of ATP by the gp44/62 complex. We demonstrate here that processive T4 holoenzyme-like DNA synthesis can be obtained without hydrolysis of ATP by simply adding gp45 to the T4 DNA polymerase at extremely high concentrations, effectively bypassing the ATPase subunits (gp44/62) of the accessory protein complex. The amount of gp45 required for the gp43-gp45 heteroassociation event is reduced by addition of the macromolecular crowding agent polyethylene glycol (PEG) as well as gp32. A chromatographic strategy involving PEG has been used to demonstrate the gp43-gp45 interaction. These results suggest that gp45 is ultimately responsible for increasing the processivity of DNA synthesis via a direct and functionally significant interaction with the T4 DNA polymerase. A corollary to this notion is that the specific role of the gp44/62 complex is to catalytically link gp45 to gp43.
Reddy
M K
MK
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Weitzel
S E
SE
von Hippel
P H
PH
D000255:Adenosine Triphosphate / Q000378:metabolism; D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003090:Coliphages / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013698:Templates, Genetic; D015534:Trans-Activators; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C060035:gp32 protein, Enterobacteria phage T4; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase