During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5' ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5' end with 32P from ,6-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released nP-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name "initiator RNA" for it. The covalent linkage of initiator RNA to 5' ends of growing DNA chains was substantiated by the finding that 3P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [a-"P]dTTP.While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5' end with 3P released a variety of different [OP]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence.Elongation of progeny strands of the DNA of polyoma or simian virus 40 is discontinuous (1, 2) and requires multiple initiation events. From studies with isolated nuclei from polyoma-infected 3T6 cells we concluded that RNA participates in the initiations (1, 3), as was previously suggested for microbial systems (4, 5). Alkaline hydrolysis of purified progeny strands synthesized from any of the four a-"2P-labeled deoxynucleoside triphosphates transferred isotope to all four ribonucleotides, demonstrating that RNA was attached to the 5' ends of growing DNA chains and that the RNA-DNA link was not unique (3). The 5' ends of RNA labeled with ['H]-GTP or [#t-"2P]GTP could be recovered as guanosine tetraphosphate (6). Furthermore, digestion of DNA progeny strands with pancreatic DNase released an RNA species that on polyacrylamide gels had the size of approximately a decanucleotide. We suggested that this might be the RNA that initiates the discontinuous synthesis of polyoma DNA (6).In the present communication we demonstrate that this "decanucleotide," for which we propose the name "initiator RNA (iRNA)," is covalently linked to the 5' ends of DNA progeny strands. It has no unique base sequence but appears to be quite homogeneous in size. MATERIALS AND METHODSAll enzymes were obtained from Worthington Biochemical Corp. Pancreatic DNase I was treated with bentonite (7) Abbreviations: pppAp and pppGp, the 5'-triphosphates, 2(3')-monophosphates of adenosine and guanosine, respectively; iRNA, initiator RNA. * This paper is no. IV in a series "Replication of Polyoma DNA in Isolated Nuclei." The preceding paper is ref. 3. 4901 and was free from RNase activity; pancreatic RNase, heated at 950 for S min before use, was free from DNase; snake venom diesterase was free from monoesterase activity (8).'H-Labeled ribonucleoside triphosphates and...

ABSTRACT2'-Deoxy-2'-azidocytidine, a nucleoside analogue, inhibits polyoma DNA synthesis. The inhibition results in a 5.5-fold decrease in the amount of replicating intermediates, as detected by electron microscopy. The results indicate that replication is inhibited at an early step, possibly in the initiation of new rounds of replication. In contrast, the effect on the elon ation of DNA chains appears to be less pronounced. As a result of the inhibition, polyoma DNA molecules at an early stage of replication accumulate in the nuclei. Upon incubation of such nuclei, DNA synthesis is induced in these molecules. The replication starts from the normal origin and elongation proceeds bidirectionally at a rate close to that observed during in vivo conditions. Analysis of polyoma DNA replication in nuclei isolated from control cells indicates that both initiation and termination of replication are impaired under in vitro conditions.The replication of polyoma DNA is bidirectional, starting from a fixed origin on the circular DNA molecule (1). The elongation process, as studied by incubation in vitro of nuclei isolated from polyoma-infected cells, occurs discontinuously by the synthesis of small fragments. ("Okazaki fragments") that are joined into longer molecules (2, 3). The Okazaki fragments are initiated by a decanucleotide of RNA (iRNA) (4) which at a later step is removed and the gaps are filled in. However, in contrast to the initiation of Okazaki fragments, only limited information exists concerning the first initiation event-i.e., the initiation of a new round of replication. One assumes that host gene products as well as the virus-coded gene A product are involved (5-7).We have earlier reported (8) that the nucleoside analogue 2'-deoxy-2'-azidocytidine (Cz) inhibits DNA synthesis in mammalian cells. Based on experiments using isolated nuclei from polyoma-infected cells, it was suggested that the inhibition was due to interference with the initiation of DNA synthesis. The present paper is an extension of these studies and confirms the hypothesis. MATERIALS AND METHODSLabeled nucleosides and nucleotides were purchased from NEN Chemicals GmbH. Cz was synthesized according to a published procedure (9). The procedures for infection of cells, isolation and incubation of nuclei, and extraction and purification of viral DNA have been described (2,3,10 To analyze the composition of the pool of viral DNA molecules, we used polyoma-infected 3T6 cells. At 23 hr after infection, when viral DNA synthesis is maximal, the cultures were divided into two sets; one set received Cz to a final concentration of 1.9 mM, and the other set served as a control. After another 4 hr, when DNA synthesis in Cz-inhibited cells was only 10% of that in control cultures, viral DNA was selectively extracted from both sets and purified (3, 10). The two DNA samples were then mounted for analysis by electron microscopy and, in order to relax the supercoiled DNA molecules, EtBr was included in the spreading mixture. Two main types of viral molecules ...

No abstract