Abstract
Synthesis of a complementary strand on the circular viral (+)-DNA of phage phiX174, coated with single-stranded DNA binding protein, is primed by the synthesis of an oligonucleotide by the primosome. Processive primosome movement on the lagging strand with the replication fork was proposed as a model for the discontinuous portion of Escherichia coli chromosome replication (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73; Arai, K., Low, R. L., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 707-711). RNA primers covalently bound to the 5'-end of a DNA chain are heterogeneous with respect to both size and nucleotide composition. The chain length of the DNA-linked RNA primers is shorter than a decanucleotide, predominantly ranging from 1 to 9 residues. The primers start with adenylate followed mainly by a purine nucleotide (Pu) at the second position suggesting that pppA-Pu is a preferred initiation sequence. The inner sequences are more heterogeneous and no consensus or preferred sequence was found beyond the third position. The size distribution of the primer is influenced by the relative concentration of ribo- and deoxyribonucleoside triphosphates; the proportion of mononucleotide (riboadenylate) primer increases upon decreasing the relative ribonucleoside triphosphate concentration. Mapping of the transition sites from RNA to DNA on HaeIII endonuclease fragments suggest they are distributed randomly and occur frequently on the phiX174 genome. These results suggest that the selection of RNA priming sites is affected by primase at the preferred sequence 3'-T-pyrimidine nucleotide-5' on the template within the DNA domain generated by the dnaB protein. These properties of RNA priming have important implications for site selection by the primosome on the lagging strand at the replication fork of the E. coli chromosome.
Highlights
Synthesis of a complementary strand on the circular cally recognized by protein n‘ [5], the prepriming proteins viral (+)-DNA of phage 4x174, coated with single- andprimaseare assembled into a multiproteinunit,the stranded DNA binding protein, is primed by the syn- primosome [6]
In order to overcome these difficulties in vivo, we took advantage of Conversion of phage 4x174 single-stranded DNA to the the availability of the purified replication system in vitro
RNA primers covalently bound to DNA were labeled holoenzyme, and single-stranded DNA binding prote(in3, 4). either by incorporation of [r-:’2P]ATPatthe 5’-endina
Summary
Ribonucleotides were mapped on the nucleotide sequences of leased by alkaline hydrolysis for PAP, pGp, PCP, and pup. These results indicated that primer RNA linked to DNA initiatespredominantly with. DNA portion was digested with pancreatic DNase I and 3’+ 5’ exonucleaseassociatedwith T4 DNA polymerase The combination of these two enzymes liberates RNA containing a3’-hydroxyldeoxyribonucleotide at the 3’-end, as well as. Isolation of the DNA-linked Primer RNA-Thereaction was stopped hy addition of EDTA and sodium dodecyl sulfate to final concentrations of 17 mM and 0.896, respectively, and size-filtered on. Treatment hv RNases-The primer RNA was treated in a 10-pl reaction mixture containing 20 mM Tris.HCI (pH 7.6) and 10 to 20 pg of oligorihonucleotides with 2 pg of pancreatic RNase or 50 units of RNase T I for 2 h a t 37 “C. Postlabelingof the 5’-endof RNA-linked DNA and the 5”hydroxyl end of DNA created by alkaline hydrolysis of RNA-linked DNA was carried out using [y-:”P]ATP and polynucleotide kinase asdescrihed [17]
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