Site selection and structure of DNA-linked RNA primers synthesized by the primosome in phage phi X174 DNA replication in vitro.

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.