Synthesis of Polyribonucleotide Chains from the 3′-Hydroxyl Terminus of Oligodeoxynucleotides by Escherichia coliPrimase

Wuliang Sun,G Nigel Godson

doi:10.1074/jbc.273.26.16358

Abstract

Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication. The sole known activity of primase is to catalyze synthesis of short RNA chains de novo. We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide. The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system. This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages. We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E. coli DNA polymerase III holoenzyme. The significance of this finding to primases generating multimeric length RNA is discussed.

Highlights

Escherichia coli primase is responsible for priming of DNA replication in E. coli chromosome [2, 3], bacteriophage ␭ [4], ␾X174 [5, 6], and G4 [7], and plasmids ColE1 [8] and pBR322 [9]
It seemed unlikely that the large products were nascent primer RNA (pRNA) because the maximum size of pRNA synthesized from G4oric by primase in the normal in vitro synthesis reaction we used is 29-nt [7]
These products were not synthesized from the oligonucleotide itself, as no products were observed when the oligonucleotide was used in the absence of G4oric

Summary

EXPERIMENTAL PROCEDURES

The 278-nt G4oric ssDNA fragment [23] was generated by EcoRI cleavage of G4oric DNA from f1R199/G4oric circular ssDNA (f1R199 containing the 278-nt G4oric sequence [24]) with complementary oligonucleotides annealed to the EcoRI cleavage sites. 0.2 pmol of 278-nt G4oric or f1R199/G4oric ssDNA template was mixed with 2.8 pmol of oligonucleotide (this ratio or higher yielded the maximum efficiency of primed synthesis) in 10 ␮l of 20 mM Tris-HCl, pH 7.5, the mixture was. The oligonucleotide-primed template was immediately added into a 50-␮l synthesis reaction that was made up of pRNA synthesis buffer (20 mM Tris-HCl, pH 7.5, 8 mM dithiothreitol, 8 mM MgCl2, and 4% sucrose), 4 ␮g of bovine serum albumin, 4.2 pmol of SSB protein (26 pmol for reaction using f1R199/G4oric), 10 pmol of primase, and different substrates. Conditions were similar with the reaction using G4oric, except that M13mp ssDNA (0.2 pmol) was the DNA template and 2 ␮g of dnaB protein (7 pmol) was added instead of SSB. The poly(rA) size marker was prepared following the method described in Ref. 31; the ladder was standardized by comparison with the 9-nt pRNA synthesized by primase

DNA Synthesis on the Primed RNA

Nuclease Digestion

RESULTS

RNA and templates

DISCUSSION