Assembly of Replicative Complexes

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DESCRIPTION (provided by applicant) The DNA polymerase III holoenzyme of E. coli is a prototypical replicative complex, exhibiting properties in common with other cellular replicases, including a high rate of processive elongation and the ability to interact with other proteins at the replication fork, establishing the communication channels necessary to coordinate the events required for efficient chromosomal replication. A key component of all cellular replicases is a multisubunit assembly of homologous proteins that require ATP to assemble a "sliding clamp processivity factor" onto primer termini. In E. coli, this function is served by the DnaX complex, DnaX3deltadelta'/khi/psi. The dna X gene of E. coli encodes two distinct products: i, the full-length translation product and gamma, a shorter protein that arises by translational frameshifting. During the next grant period, we will study i) the assembly pathway of DNA polymerase III holoenzyme, with emphasis on the role of Pol III in steering the assembly pathway to permit a unique arrangement of DnaX subunits, ii) determine the changes in macromolecular interactions that occur during loading of the beta2 sliding clamp onto primed DNA by the DnaX complex 'clamp loader,' iii) determine the mechanism of ATPgammaS-assisted initiation complex formation that proceeds without nucleotide hydrolysis and iv) study the chaperone-like properties of DnaX in initiation complex formation.