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Escherichia coli primase primer synthesis activity characterization
Abstract
Primase, the dnaG product from Escherichia coli, is the RNA polymerase responsible for the initiation of Okazaki fragments in vivo. This makes primase a protein of central importance in DNA replication. Previous studies of primase activity have indicated that both auxiliary proteins and DNA secondary structure are required for primase activity. My goal was to define the minimum template and protein requirements for primase activity and standardize a simplified assay that could be used to study primase in depth. Results presented here demonstrate that primase, in the absence of any auxiliary proteins, was able to prime any single-stranded DNA template that contained the CTG trinucleotide sequence. Kinetic analysis of this system indicated that primase binding to single-stranded DNA templates was a weak interaction. Primase binding and/or initiation of primer synthesis appeared to be the limiting step since primers shorter than eight nucleotides in length were not identified. This suggested that in vivo primase must interact with other proteins in order to bind to and initiate primer synthesis at the proper time and place. For Okazaki fragment synthesis to occur in coordination with leading strand DNA replication, primase must be able to produce primers when necessary. This may explain the loose substrate specificity observed for primase in these studies. Primase was able to incorporate ribonucleotides, deoxyribonucleotides, and dideoxyribonucleotides into primers and, given enough time, primase could also initiate primer synthesis at sequences other than CTG. Primase also showed very low fidelity during synthesis. Given the choice to either discontinue primer synthesis and dissociate from the template or incorporate a mispaired nucleotide, primase incorporated a mispaired nucleotide approximately 5% of the time. This does raise the possibility of replication errors, if primase were to misincorporate several deoxyribonucleotides, they may not be excised by the normal prime excision mechanisms. A preliminary investigation into an exonuclease associated with primase that may serve a proofreading function is also reported.
Subject Area
Biochemistry
Recommended Citation
Swart, John Randall, "Escherichia coli primase primer synthesis activity characterization" (1994). ETD collection for University of Nebraska-Lincoln. AAI9519552.
https://digitalcommons.unl.edu/dissertations/AAI9519552