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A study of the error -prone nature of primase from Escherichia coli: The effect of DnaB helicase
Escherichia coli primase is a ssDNA-dependent RNA polymerase that initiates DNA replication by synthesizing a small RNA polymer or primer. Primer RNA is synthesized once to initiate leading strand DNA synthesis and repeatedly on lagging strand ssDNA to initiate Okazaki fragment synthesis. Previous studies have indicated that primase, acting alone, utilizes the trinucleotide sequence 5′-d(CTG)-3′ during initiation of RNA primer synthesis. Kinetic studies have also indicated that primase possesses a slow synthetic rate in vitro, which is in contrast to the high synthetic rote observed in vivo. One of the many proteins that interact with primase at the replication fork in E. coli is DnaB helicase. Many studies have shown that DnaB helicase profoundly affects the activity of primase. Given this, my goal was to determine the effect of DnaB helicase on the initiation specificity of primase. I have also examined the types of primers generated in the presence and the absence of DnaB helicase. ^ Results presented here demonstrate that primase has a high initiation specificity for the d(CTG) trinucleotide only in the absence of DnaB helicase. The initiation trinucleotide preference is greatly relaxed in the presence of DnaB helicase such that initiation will occur from nearly every trinucleotide. This has a very significant biological implication in Okazaki fragment initiation on the lagging strand. ^ Primase, in the absence of any accessory proteins, generated two different types of primers. The majority of the primers synthesized by primase were longer than the template and were named “overlong primers”. In contrast, the helicase-stimulated RNA primers were template length-dependent, and ranged from 10 to 16 nucleotides in length. The study presented here demonstrates that the initiation sites of the two classes of primers are different. DnaB helicase stabilized the open or single-stranded conformation of the template that resulted in the synthesis of the template length-dependent primers. In the absence of DnaB helicase, primase stabilized the 3′-end hairpin conformation of the template so that the template's 3′ -hydroxyl served as a “DNA primer” from which primase elongated to create “overlong primers”. ^ The effect of metal ions in primer synthesis has also been studied. Most of the metal ions tested in the “simple” primer synthesis assay can support primer synthesis but only in the presence of DnaB helicase. It was observed that the activity of primase was stimulated several-fold in the presence of Mn+2, both in the presence and the absence of DnaB helicase. The increased activity appeared to be partly due to the efficient binding of primase to the ssDNA template. Mn+2 also affected the fidelity of primase. ^
Bhattacharyya, Saumitri, "A study of the error -prone nature of primase from Escherichia coli: The effect of DnaB helicase" (2000). ETD collection for University of Nebraska - Lincoln. AAI9962054.