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Bacterial DNA replication enzymes: The interaction between primase and helicase

Christopher A Frey, University of Nebraska - Lincoln

Abstract

Some of the least well understood aspects of DNA replication are the mechanisms of activity for primase and helicase. In understanding the mechanisms of activity for these enzymes, it is important to characterize the interaction between these enzymes because this interaction is responsible for initiation of the synthesis of new DNA strands. Work done by Corn et al [Corn, J.E., et al. (2005) Mol. Cell 20, 391-401] suggested many intricacies of this interaction and led to the proposal of a model for this activity. This proposed model is termed the crosstalk model wherein primase uses helicase as a platform for a primase-primase interaction to initiate priming activity. Alternatively, there is an entropic model that proposes that the most important part of the primase-helicase interaction is to bring primase into the replication fork such that it can access free single-stranded DNA. ^ Addressed first is the possibility that primase may bind DNA or function as a dimer. Primase from S. aureus, E. coli, and G. stearothermophilus were tested to try to detect the presence of a dimeric state in primase by chemical crosslinking or sedimentation equilibrium. The primase concentrations used were similar to those used for standard primase activity assays. Primase was determined to be monomeric under all conditions used, including in the presence of ssDNA. ^ To help understand the interaction between primase and helicase, chimeras and truncated constructs of the two enzymes from G. stearothermophilus were created. Quaternary structural characterization of these chimeric constructs verified that the p33 domain of helicase was responsible for hexamerization but the p17 domain helps control the formation of the complex such that a heptamer is formed in the absence of the p17 domain. The other unusual finding is that chimeras with the p33 aggregate at the optimal growing temperature for G. stearothermophilus which suggests there are other components in vivo that help stabilize the helicase. ^ To further investigate the models of primase-helicase interaction, sedimentation velocity and mass spectrometry were used to investigate the stoichiometry of primase-helicase complexes. The activity of primase when stimulated by helicase was also tested using a sensitive fluorometric assay. This confirmed that a 3:1 primase to helicase complex assembles such that it is the only detectable complex formed. The activity data confirm that the primase acts in a cooperative manner. The results show that the crosstalk model is a valid model, but the stimulation of primase by helicase is less than 5-fold. This low level of stimulation suggests that an entropic model might be a better explanation for the primase-helicase interaction than the crosstalk model. ^

Subject Area

Biology, Molecular|Chemistry, General|Chemistry, Biochemistry

Recommended Citation

Frey, Christopher A, "Bacterial DNA replication enzymes: The interaction between primase and helicase" (2013). ETD collection for University of Nebraska - Lincoln. AAI3559683.
http://digitalcommons.unl.edu/dissertations/AAI3559683

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