Dissociative properties of the proteins within the bacteriophage T4 replisome.
DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5' to 3' direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed.
D000251:Adenosine Triphosphatases / Q000737:chemistry; D017122:Bacteriophage T4 / Q000201:enzymology*; D017123:Bacteriophage T7 / Q000378:metabolism; D002151:Calorimetry; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005799:Genes, Dominant; D008956:Models, Chemical; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013698:Templates, Genetic; D013997:Time Factors
C022262:Okazaki fragments; D004247:DNA; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases