Enzyme studies of replication of the Escherichia coli chromosome

Progress of the replication forks of the Escherichia coli chromosome depends on a multisubunit DNA polymerase (for chain elongation) and a primosome (for chain initiations), together comprising about 30 polypeptides with a mass in excess of 10(6) daltons. Integration of their actions with those of helicases and DNA binding proteins suggest a more complex and integrated replisome assembly with novel possibilities for concurrent replication of both parental strands. Initiation of a new cycle of chromosome replication at its unique 245-bp (oriC) is being studied in a soluble enzyme system with plasmids, autonomous replication of which depends on the oriC sequence. Required proteins include RNA polymerase, DNA gyrase, dnaA protein (with 4 strong binding sites in oriC), HU protein, and additional proteins (e.g., topoisomerase I and ribonuclease H) that confer oriC specificity by suppressing initiation of replication elsewhere on the duplex DNA. Clarification of the biochemical mechanisms of replication is fundamental for understanding cell growth and development. Knowledge of the biochemistry of initiating a cycle of chromosome replication opens the way toward exploring the regulation of the cell cycle. I remain faithful to the conviction that anything a cell can do, a biochemist should be able to do. He should do it even better, being freed from the constraints of substrate and enzyme concentrations, pH, ionic strength, and temperature, and by having the license to introduce novel reagents to drive or restrain a reaction. Put another way, one can be creative more easily with a reconstituted system. One can grapple directly with the molecules instead of trying by remote means to manipulate their structures or levels in the intact cell. Enzyme purification carries many dangers beyond the well-known exposure of the fragile enzyme to the hostilities of an unfamiliar environment, high dilution, glass containers and a denaturable investigator. But the rewards of enzyme purification have justified the effort. The polymerases, nucleases, ligases purified out of curiosity about the mechanisms of replication, repair and recombination have supplied the cast of actors responsible for the current drama of genetic engineering. Beyond the uses of these enzymes as reagents, understanding the mechanisms of DNA metabolism will have practical value in manipulating the replication of plasmids and viruses and the expression of their genes and, beyond that, in obtaining a more secure grasp of chromosome structure and function.