In this application, studies are proposed to investigate the regulation of DNA replication at two levels- (i) the allosteric regulation that occurs between the subunits of the DNA polymerase III holoenzyme and the primosome to permit coordination of the multiple reactions that occur at the replication fork; and (ii) the genetic regulation of replication protein synthesis. Replication at the fork requires the coordinated action of the 10 subunits of the DNA polymerase III holenzyme with the even-protein primosome-primary complex on a template coated with single-stranded DNA binding protein. These coordinate reactions include; (i) signalling between the primase as it synthesizes a primer for a new Okazaki fragment and the lagging strand half of holoenzyme, causing it to release and cycle to the new primer; (ii) communication between the holoenzyme and primase, causing it to limit the length of primer synthesis to ca. 10 nucleotides; (iii) communication between the holoenzyme and the DnaB helicase in the primosome, stimulating its activity ca. 15-fold an presumably linking these two complexes ina coupled reaction;' and (iv) linkage of the leading and lagging strand halves of the holoenzyme, sequestering the lagging strand polymerase at the replication fork so that it does not reequilibrate with solution after release from a completed Okazaki fragment. We will identify specific subunit-subunit contacts that enable these communication links using surface plasmon resonance and other biophysical techniques. We will then identify the assembly state and the effectors that attenuate contacts, focusing primarily on identifying the holoenzyme subunits that contact primase and testing the hypothesis that a tau interaction with DnaB is required for stimulation of its helicase activity. We will also assemble aberrant dimeric replication complexes to dissect the contributions of tau- pol III contacts from those caused by dimerization of the enzyme by tau. Studies of the genetic regulation of replication protein synthesis will focus on the expression of dnaE, the structural gene for the polymerase catalytic subunit alpha. The dnaE gene resides in a complex operon with four genes involved in membrane biogenesis and rnhB, a second RNAseH. Translational coupling of the first five genes of the operon including dnaE is suggested from the sequence and our preliminary results. We have identified the 5'-ends of nine transcripts that express dnaE, indicating complex regulation. Two transcript 5'-ends, PB and PC, are generated by cleavage with RNase III. Based on the finding that both 3'- and 5'ends map to PR, we hypothesize that it is also a processing site, cleaved by an as yet undiscovered riboendonuclease. Studies will focus on determination of whether PR is a processing site or a coincident transcription terminator and promoter, determination of the importance of translational coupling and RNase processing on the expression of dnaE, and determination of the principal factors that regulate dnaE expression.
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