RNA PRIMERS IN SIMIAN-VIRUS 40 DNA-REPLICATION .2. DISTRIBUTION OF 5' TERMINAL OLIGORIBONUCLEOTIDES IN NASCENT DNA

[摘要] A cell-free SV-40 DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages copurified with SV-40 replicating DNA. These linkages were identified by transfer of a fraction of the 32P from the 5'' position of a deoxyribonucleotide to 2''(3'')rNMP [ribosomal nucleoside monophosphate] upon either alkaline hydrolysis or RNase T2 digestion of SV-40 replicating [32P]DNA. Alkaline hydrolysis also exposed 5'' terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5'' termini (RNA primers). The density of a portion of the Okazaki pieces in KI gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of RNase T2 or KOH converted about 1/3-1/2 of them into a 2nd well defined group of DNA chains of greater electrophoretic mobility in polyacrylamide gels. The increased mobility corresponded to the removal of at least 7 residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that 1/3-1/2 of them contained rN-32P-dN linkages, the oligoribonucleotides must be covalently attached to the 5'' ends of nascent DNA chains. Although the significance of 2 populations of Okazaki pieces, 1 with and 1 without RNA primers, is not perfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers. Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in ascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5'' end of growing daughter strands. 32P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV-40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. The positions of RNA primers are not determined by a specific set of nucleotide sequences.