Discontinuous DNA replication of Drosophila melanogaster is primed by octaribonucleotide primer.

Abstract

To investigate the precise structure of eucaryotic primer RNA made in vivo, short DNA chains isolated from nuclei of Drosophila melanogaster embryos were analyzed. Post-labeling of 5' ends of short DNA chains with polynucleotide kinase and [_Y-32P]ATP revealed that 7% of the DNA fragments were covalently linked with mono-to octaribonucleotide primers at their 5' ends. Octaribonucleotides, the major component (ca. 30%), formed the cap structure in the reaction with vaccinia guanylyltransferase and [o-32P]GTP, indicating that they were the intact primer RNA with tri-(or di-) phosphate termini, and the shorter ribooligomers were degradation intermediates. The intact primers started with purine (A/G ratio, 4:1), and the starting few ribonucleotide residues were rich in A.Primer RNA molecules for discontinuous DNA replication of eucaryotic genomes were extensively studied by using isolated nuclei of papovavirus-infected and noninfected mammalian cells (4,8,18,28,29). Chain length of the primer RNA was approximately decanucleotide, synthesis of the primer was started with pppA or pppG, and nucleotide sequence at the RNA-DNA junction was random (18). Recently, DNA primase activities associated with purified DNA polymerase a of various eucaryotic systems were found, and the size of primer RNA was approximately decanucleotide in most cases (1, 2,9,22,26). Primer RNA molecules in vivo were analyzed by using a human lymphoblastoid cell line (27) and a simian virus 40-infected monkey cell line (6). Their chain length varied from mono-up to approximately nonanucleotide, and it was suggested that the longest primer molecules probably corresponded to the primary products, whereas the shorter molecules were in various stages of excision process.The purposes of this work were to detect intact primer RNA molecules in eucaryotic cells, to determine their exact sizes, and to clarify whether any characteristic nucleotide sequences were present. Rigorous investigations on these points have not been done previously with eucaryotic primer RNA made in vivo. Early embryos of Drosophila inelanogaster are used for the analyses because their rapid rate of nuclear division suggests an abundance of nascent chains (10,17 A residues, though lesser so than the in vitro primer (32), and the 3'-terminal region is rich in A and U residues. These results raise the possibility that primer synthesis starts preferentially on a T-rich region on the template strands. MATERIALS AND METHODSReagents and enzymes. 32P, (carrier-free), G(5')pppA and G(5')pppG, nitrocellulose, nuclease P1, nuclease SW, and vaccinia guanylyltransferase (capping enzyme) have been described previously (32). Pancreatic DNase I, T4 polynucleotide kinase, Escherichia coli alkaline phosphatase, and T4 DNA polymerase have been described previously (15).[_-32PJATP and [a-3 PJGTP were prepared as described previously (32). Dihydroxyboryl Bio-Gel P-60 (boronate gel) was prepared by the method of Okayama et al. (16).Purification and terminal labeling of short DNA fragments. D. melanogaster...

Eucaryotic primase

“…The 5' residue is also a purine (A: G ration 2 4). Sequence analysis of the SV40 primers showed that discontinuous DNA replication occurs exclusively on the lagging strand relatively to the origin of the viral genome [34, 351. More recently, the size and the composition of primers isolated from Drosophilu embryos have been investigated [36]. The authors used vaccinia guanylyltransferase to label intact nascent DNA fragments having a 5'-di-or triphosphate group to discriminate them from partially hydrolyzed primers.…”

“…Yoda and Okazaki [53] demonstrated that primase activity in a cytoplasmic extract of D. melanogaster embryos synthesizes hexa-to heptaribonucleotides. Primers synthesized in vitro could be one or two nucleotides shorter than their counterparts in vivo [36,531. Primers synthesized on homopolymers in the absence of the complementary dNTP are longer.…”

“…This possibility is raised by the fact that only 30% of the isolated primers are octaribonucleotides in Dlosophila embryos. This proportion decreases to about 2%) only when all the nascent fragments are considered, including those bearing no RNA [36]. The fact that most of the initiator RNAs (about 70%) are obtained as a broad family of chains ranging in size from one to seven nucleotides [33, 361 suggests a rapid and progressive removal of the primer and/or a quick degradation during isolation.…”

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

“…As explained for mSVO2, the combined data revealed that the lengths of nascent chains derived by a direct comparison with the lengths of sequenced DNA chains had to be shortened by 6.5 bases in the case of mSVO9 and 7 bases in the case of mSVO7 to locate the actual initiation sites. Positions of 15 major sites on mSVO9 DNA and 14 major sites on mSVO7 DNA are provided in Fig. 4.…”

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

“…Although little is known about the molecular mechanisms that regulate entry of eucaryotic cells into the S phase, substantial genetic and biochemical evidence (reviewed in references 7, 10, and 14) suggests that replication is initiated at specific sites (or origins of replication) and proceeds bidirectionally (10) in a semidiscontinuous fashion (19). Selection of those replicons slated to be synthesized immediately upon entry into the S phase may be regulated at the level of either initiation or elongation, perhaps by the interaction of trans-acting initiation factors with specific origin sequences or by differential elongation of performed initiation complexes.…”