The dynamic processivity of the T4 DNA polymerase during replication.
The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of approximately 0.0013 sec(-1) was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.
Yang
Jingsong
J
Department of Chemistry, 414 Wartik Laboratory, Pennsylvania State University, University Park, PA 16802, USA.
Zhuang
Zhihao
Z
Roccasecca
Rosa Maria
RM
Trakselis
Michael A
MA
Benkovic
Stephen J
SJ
D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism; D004247:DNA; D004261:DNA Replication; D004270:DNA, Circular; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D013698:Templates, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D004270:DNA, Circular; D004279:DNA, Viral; D046911:Macromolecular Substances; C022262:Okazaki fragments; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D004259:DNA-Directed DNA Polymerase