Abstract
In the presence of dATP, glycerol, and Tris buffer, the DNA primase isolated from Thermococcus kodakaraensis catalyzed the formation of dAMP and two products that were identified as dAMP-glycerol and dAMP-Tris. These products were formed by the T. kodakaraensis p41 catalytic subunit alone and the T. kodakaraensis p41-p46 complex in the absence of a DNA template. They were not formed with preparations containing the catalytically inactive p41 subunit. Similar glycerol and Tris derivatives as well as dNMPs were also formed with dGTP, dCTP, or dTTP. The mechanism contributing to the formation of these products and its implications in the initiation reaction catalyzed by the T. kodakaraensis primase are discussed.
Highlights
Archaeal primase catalyzed the DNA-independent synthesis of oligonucleotide-like products from dNTPs
In the accompanying article [1], we noted the formation of two 32P-labeled products following incubation of the T. kodakaraensis primase complex or the T. kodakaraensis p41 catalytic subunit with [␣-32P]dNTPs that migrated as 32P-labeled ϳ16and ϳ25-nt bands following urea-PAGE separation
We examined whether RNA and DNA synthesis catalyzed by the T. kodakaraensis primase complex as well as the p41 subunit was affected by the presence or absence of glycerol and/or Tris
Summary
Archaeal primase catalyzed the DNA-independent synthesis of oligonucleotide-like products from dNTPs. In the accompanying article [1], we noted the formation of two 32P-labeled products following incubation of the T. kodakaraensis primase complex or the T. kodakaraensis p41 catalytic subunit with [␣-32P]dNTPs that migrated as 32P-labeled ϳ16and ϳ25-nt bands following urea-PAGE separation. Their formation required a catalytically active p41 subunit but was independent of a DNA template. We show that each of the four common dNTPs support formation of these products, whereas rNTPs are considerably less active In addition to these derivatives, dNMP is ganic pyrophosphatase; nt, nucleotide; TE, Tris-EDTA; dAp, 3Ј-deoxyadenosine phosphate; pdAp, 5Ј,3Ј-deoxyadenosine diphosphate. The mechanism governing formation of these products is unclear, we discuss its possible significance in the DNA primase initiation and aborted initiation-elongation reactions
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