9303921 O'Donnell The polymerase that replicates the chromosome of E. coli, DNA polymerase III holoenzyme (polIII), consists of 10 different subunits. As a holoenzyme, polIII hydrolyzes ATP to bind tightly to DNA enabling highly processive DNA synthesis. The holoenzyme can be dissociated into subassemblies: the 3-subunit core polymerase @ subunit (polymerase), @subunit (3'-5' exonuclease) and 0 subunit , the 5-subunit y complex (y@@xy subunits); the B subunit and the t subunit. The function of the y complex is to couple ATP to deliver the B subunit to DNA. The B subunit is a dimer shaped like a ring (X-ray analysis) and completely encircles DNA. The B ring then binds the core polymerase acting to tether it to DNA for highly processive synthesis. The t subunit dimer binds two core polymerases together, presumably for simultaneous synthesis of both leading and lagging strands of the duplex chromosome. Synthesis of the lagging strand is discontinuous, being composed of approximately 300 fragments (Okazaki fragments). Hence, each time polIII on the lagging strand completes an Okazaki fragment it must be capable of rapidly recycling itself from the end of the fragment to initiate synthesis of new fragment. However, the polIII is bound to DNA so tightly by the B subunit ring that it remains tightly bound to a completed product DNA rather than cycling to new primed templates. We have recently discovered a mechanism whereby polIII rapidly cycles to new DNA templates. The mechanism entails a novel disaggregation of the polIII structure followed by reassembly in which the polIII disengages its B ring specifically upon completing the replication of a DNA template and then it reassembles with a B ring on a new DNA molecule. This proposal aims to discover the detailed molecular basis behind this polymerase transfer event. New reagents and technology important to the proposed work include large amounts of pure preparations of each of the 10 subunits of polIII an d the ability to reconstitute the entire holoenzyme from these individual proteins. Hence we propose to perform a series of subunit omission studies to determine which subunits are responsible for disengaging the B ring from polIII and which are needed to transfer the polymerase to the next B ring on another DNA molecule. We will also investigate how the B rings that are left on DNA as the polymerase cycles to multiple templates, are themselves removed from the DNA for eventual reutilization. Then we will develop a replication fork system to investigate which subunits of polIII are needed for efficient interaction with the helicase and primase during ongoing synthesis of both strands of duplex DNA. %%% The genetic material is in the form of two long interwound strands of deoxyribonucleic acid, or DNA. These two DNA strands contain the information needed to instruct the cell how to live (eg. eat and divide) and therefore they must be duplicated prior to cell division such that each new cell receives a copy of these instructions. This process of DNA duplication is called "replication" and it is performed in a series of complicated steps many of which have yet to be precisely defined. Each step is performed by a different protein molecule and therefore several "replication proteins" are needed to duplicate DNA. These several replication proteins assemble together to form a multiprotein complex in which each protein occupies a distinct position where it can carry out its individual function in the overall process. The multiprotein complex is analogous to a machine in which each gear performs a function, except here the gears are proteins and each protein performs a function. Recently this laboratory showed that one of the gears of this "replication machine" of the bacterium, Escherichia coli, is a protein shaped like a washer (ie. a ring) and completely encircles the strands of DNA for the purpose of tethering the replication machine down to DNA so it can eff iciently perform its function. Each of the replication proteins for this bacterium are now available in separate test tubes and we have learned how to assemble the machine from these separate gears. With these individual protein reagents and the knowledge of how they assemble into a machine, this proposal aims to determine what the function is of other individual proteins, or gears, of this replication machine as it duplicates the two strands of DNA. ***
