Polymerases and the Replisome : Review Machines within Machines Polymerases : Template - Directed Phosphoryl Transfer Machines Synthesis of the new DNA strands occurs as a result

of a collaboration between the synthetic capacities of Massachusetts Institute of Technology multiple polymerases. Two types of polymerases are Cambridge, Massachusetts 02139 required: primases, which start chains, and replicative polymerases, which synthesize the majority of the DNA (Kornberg and Baker, 1992). The replication fork, how-Synthesis of all genomic DNA involves the highly coordi-ever, contains at least three distinct polymerase activi-nated action of multiple polypeptides. These proteins ties: a primase and a replicative polymerase for each of assemble two new DNA chains at a remarkable pace, the two template strands. In E. coli, primase is a single approaching 1000 nucleotides (nt) per second in E. coli. polypeptide, and the replicative polymerase is a dimer If the DNA duplex were 1 m in diameter, then the follow-of DNA polymerase (pol) III core and several accessory ing statements would roughly describe E. coli replica-proteins that together form the pol III holoenzyme (re-tion. The fork would move at approximately 600 km/hr viewed in Marians, 1992; Kelman and O'Donnell, 1995). (375 mph), and the replication machinery would be about Similarly, phage T4 has one primase and one replicative the size of a FedEx delivery truck. Replicating the E. coli polymerase that appears to function as a dimer (Alberts, genome would be a 40 min, 400 km (250 mile) trip for 1987; Munn and Alberts, 1991). The situation in eukaryo-two such machines, which would, on average make an tic cells is slightly different (Stillman, 1994). The primase error only once every 170 km (106 miles). The mechanical is in a tight complex with a DNA polymerase (pol ␣) and prowess of this complex is even more impressive given eukaryotic cells have two distinct replicative polymer-that it synthesizes two chains simultaneously as it ases: polymerase ␦ (pol ␦) and polymerase ⑀ (pol ⑀). moves. Although one strand is synthesized in the same All the replicative polymerases have one large subunit direction as the fork is moving, the other chain (the that contains the polymerase active site and, with the lagging strand) is synthesized in a piecemeal fashion exception of pol ␣–primase, the same subunit or an (as Okazaki fragments) and in the opposite direction of associated polypeptide carries a proofreading 3Ј→5Ј ex-overall fork movement. As a result, about once a second onuclease. The polymerase subunits also interact with one delivery person (i.e., polymerase active site) associ-proteins that dramatically influence their association ated with the truck must take …