| Abstract: |
Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the laggingstrand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage øX174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a øX174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3′ →5′ exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5′→3′ DNA polymerase activity nor the 3′→5′ exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus. |
| Keywords: |
controlled study; dna binding protein; nonhuman; dna polymerase; dna replication; dna synthesis; protein protein interaction; enzyme activity; rna; dna; escherichia coli; dna, viral; dna, single-stranded; exonuclease; dna, bacterial; dna template; dna primase; dna polymerase iii; rna nucleotidyltransferases; bacteriophage phi x 174; priority journal; article; unidentified bacteriophage; electrophoresis, agar gel; support, u.s. gov't, p.h.s.
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