
Charles Clifton Richardson, M.D.
Fig. 1A major goal is to define, in molecular terms, the mechanism by which a chromosome is replicated. The replication of the chromosome of bacteriophage T7 has been used as a model system. T7 has evolved an efficient and economical system for DNA replication. The cartoon depicts the T7 replisome and illustrates the major molecular motors and contacts. On the leading strand, T7 DNA polymerase (gp5) undergoes multiple conformational changes as it moves from one template position to another and senses the correct fit of an incoming deoxyribonucleoside triphosphate. E. coli thioredoxin (trxbinds tightly to the thumb subdomain of gp5 and increases its processivity 100-fold. The interaction with gp5 also creates docking sites for the other T7 replication proteins. Unwinding of the DNA to create a ssDNA template for gp5/trx is accomplished by the helicase located in the C-terminal half of T7 gene 4 protein (gp4). Gp4 assembles as a hexamer on the lagging strand and uses the energy of hydrolysis of dTTP to translocate 5’ – 3’ on ssDNA; it unwinds duplex DNA that it encounters. The assembly of the hexamer creates six NTP-binding sites at the interface of the subunits; nucleotide hydrolysis results in conformational changes that are conveyed to the central core through which the DNA passes.
In order for the lagging strand to be replicated it is necessary to periodically synthesize an RNA primer. Primer synthesis is catalyzed by the primase located in the N-terminal half of gp4. The tetraribonucleotides, synthesized at specific DNA sequences, are extended by a lagging strand polymerase to generate Okazaki fragments several thousand nucleotides in length. The lagging strand folds back on itself such that the lagging strand DNA polymerase can interact with the helicase. This association of the two polymerases enables both strands to be synthesized in the same overall direction and synthesis of both strands now proceeds at identical rates. The folding of the lagging strand creates a replication loop of lagging strand DNA that contains the nascent Okazaki fragment. These loops, visualized by electron microscopy or real time single molecule techniques, form, grow, and resolve, to generate Okazaki fragments of uniform lengths of around 1000 nucleotides. Upon initiation of primer synthesis the helicase halts movement. This brake mechanism is dependent on the interaction of the primase with the helicase.
Although proteins in the T7 replisome are relatively few, there are a large number of interactions that provide for the molecular motors and switches. All of the individual proteins have been crystallized and their crystal structures determined. In addition, a snapshot of an actively synthesizing polymerase is available in the crystal structure of gp5/trx in complex with a primer-template and a dNTP. The structure of the full length hexameric gp4 and those of the helicase and primase domains provide insight into the communication between the domains as well as the mechanism of translocation of the helicase on DNA.
In addition to these studies on the replisome studies are designed to understand the acquisition of host functions by T7. T7 derives the nucleotide precursors for DNA synthesis from the breakdown of the host DNA. T7 gene 1.7 protein is a nucleotide kinase that converts dTMP and dGMP to dTDP and dGDP that are then converted to the dNTP by host kinases. E. coli adenylate kinase and dCMP kinase account for the formation of dADP and dCDP. T7 gene 1.2 protein inhibits an E. coli dGTPase that hydrolyzes dGTP to dG and tripolyphosphate. Gene 2 protein binds to the Beta subunit if E. coli RNA polymerase and inhibits its activity, a prerequisite for packaging of the DNA.
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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Phys Rev Lett
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