RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) 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 were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 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 potassium iodide 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 either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half 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 two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly 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 nascent 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 SV40 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. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Mechanism of DNA chain growth: XV. RNA-linked nascent DNA pieces in Escherichia coli strains assayed with spleen exonuclease

A new method for the detection and assay of RNA-linked nascent DNA pieces has been developed. The method relies on selective degradation by spleen exonuclease of radioactive 5′-OH terminated DNA produced from the pulse-labelled nascent pieces upon alkaline hydrolysis. Analysis with this method in wild type Escherichia coli has shown relatively high proportions of the RNA-linked molecules after shorter pulses and in the smaller pieces, supporting the transient nature of the RNA attachment to the nascent pieces. The RNA-linked nascent DNA pieces are accumulated by both E. coli polAex1 (defective in 5′ → 3′ exonuclease of DNA polymerase I) and E. coli polA12 and polA1 (defective in polymerase of DNA polymerase I), suggesting the requirement of the concerted action of both 5′ → 3′ exonuclease and polymerase of DNA polymerase I for the removal of the RNA attached to the nascent pieces. Most of the nascent DNA pieces accumulated by E. coli ligts7 (defective in DNA ligase) are not linked to RNA, as expected from the direct role of DNA ligase in joining of the pieces. The analysis also has shown that a large portion of the nascent DNA pieces present in the cell under the normal steady-state conditions are not linked to RNA and that the level of the RNA-free DNA pieces is also increased in polA mutants. These findings suggest that the removal of RNA from the nascent pieces is a relatively rapid process and the joining reaction is a rate-limiting step that requires the concurrent action of DNA polymerase and DNA ligase.     

Assay of RNA-linked nascent DNA pieces with polynucleotide kinase.

The 5′-OH end of DNA created upon alkaline hydrolysis of the RNA-linked nascent DNA pieces can be labeled with [γ-³²P]ATP using T4 polynucleotide kinase. However, it is difficult to use this method for the assay of these molecules in the presence of RNA-free DNA pieces because of the exchange reaction between the γ-phosphate of ATP and the 5′-phosphate of DNA catalyzed by the kinase. This difficulty can be circumvented by performing the polynucleotide kinase reaction at 0°C, where little exchange reaction occurs. Using these conditions, $\text{E. coli pol}$Aexl, a mutant defective in the 5′ → 3′ exonuclease activity of DNA polymerase I, is shown to contain several times as many RNA-linked DNA pieces as the wild type.     

Initiator RNA of nascent DNA from animal cells.

Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ-³²P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines ($\text{A}\text{G}\text{=}\text{3·5}\text{1}$), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells.     

RNA-linked nascent DNA pieces in phage T7-infected Escherchia coli. II. Primary structure of the RNA portion.

Short DNA chains were purified from phage T7 infected E. coli cells and 5' ends were labeled with 32P. By an alkali-treatment, pNp's rich in pAp and pCp were liberated from the T7 short DNA chains. After digestion of the [5'-32P] short DNA with the 3' to 5' exonuclease of T4 DNA polymerase, [5'-32P] mono- to pentaribonucleotides tipped with a deoxyribonucleotide residue at their 3' ends were isolated. 5' terminal ribonucleotides were; exclusively AMP in the penta- and the tetraribonucleotides, mostly CMP in the triribonucleotide and mainly CMP and AMP in di- and monoribonucleotides. The 5' terminal dinucleotide of the penta- and the tetraribonucleotides was pApC. The nucleotide sequence of the tetraribonucleotide was mainly pApCpCpN and some pApCpApN, where N was mainly A and C. These results indicate that oligoribonucleotides shorter than trinucleotide may result from in vivo degradation of the tetra- and pentaribonucleotides. A possibility that the tetra- and pentaribonucleotides with a 5' triphosphate terminus are the intact primers for the discontinuous T7 DNA replication is discussed.     

Evidence for the presence of ribonucleotides at the 5' termini of some DNA molecules isolated from Escherichia coli polAex2.

We have purified a set of small DNA molecules from various strains of exponentially growing Escherichia coli, including E. coli polAex2. This material included very short molecules (2 S), the nascent DNA (“Okazaki fragments”) and some longer molecules. Most of the [³H]thymidine incorporated during a brief period of labeling was found in the 5 S to 15 S Okazaki fragments. There was a large number of the 2 S molecules in the cell. The properties of the 5′ ends of these molecules were investigated using three procedures. (1) The DNA preparation, pulse-labeled with [³H]thymidine, was reacted with polynucleotide kinase and ATP to insure that all 5′ ends were phosphorylated. After subjection of the DNA to alkaline hydrolysis, the proportion of incorporated ³H pulse-label that became susceptible to digestion by spleen exonuclease was determined. In different experiments there was an increment of up to 20% in the amount of pulse-labeled E. coli polAex2 DNA that could be hydrolyzed by the exonuclease after treatment with alkali. (2) As in the preceding protocol, phosphorylation of the 5′ ends was assured by reaction with kinase and ATP; the preparation was then treated with alkali and the number of 5′-OH ends generated that could be labeled with ³²P using [γ-³²P]ATP and kinase in a second reaction was determined. The data indicated that 3 to 30% of the molecules could be labeled after alkali digestion, but not before. (3) The DNA molecules were reacted with kinase and [γ-³²P]ATP after having been exposed previously to alkaline phosphatase. The end-labeled molecules were then subjected to an alkaline hydrolysis and the resulting hydrolysate chromatographed on a polyethyleneimine-cellulose thinlayer plate. Alkali treatment was found to release 2′(3′),5′-ribonucleoside diphosphates from 1 to 30% of the molecules; pAp and pGp predominated. Control experiments showed that these ribonucleotides were covalently linked to the 5′ ends of polydeoxyribonucleotides. Curiously, the smaller the DNA molecule the less likely it was to possess a 5′-terminal ribonucleotide. Very few apparent RNA/DNA molecules were observed in the non-polAex2 strains tested. These observations are in part in agreement with previous reports, and we infer that at least some of the nascent E. coli polAex2 DNA molecules are initiated in vivo with a ribonucleotide primer. The relatively smaller proportion of molecules with apparent 5′-terminal ribonucleotides among the smaller DNA molecules and in strains other than E. coli polAex2 suggests to us that there may exist a mechanism for initiating DNA molecules that does not require an RNA primer.     

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

Summary The presence of RNA-linked nascent DNA pieces in T7 phage-infectedEscherichia coli cells has been shown by the selective degradation of the 5-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43°C, the proportion of RNA-linked DNA pieces in nascent short DNA is 50 to 60% in T7ts136 (ts mutant of gene 6) phage-infectedE. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7ts136-infectedE. coli temperature sensitivepolA mutants at 43° C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.     

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Alkali-labile structures linked to the 5'' ends of Bacillus subtilis short DNA chains.

Alkali-labile portion covalently linked to the 5' ends of Bacillus subtilis short DNA chains, the putative primer RNA for discontinuous DNA synthesis, was isolated and analyzed using a temperature sensitive DNA polymerase I mutant, which accumulates nascent DNA fragments at a restrictive temperature. A novel oligonucleotide structure as well as mono- to triribonucleotide stretches were isolated at the 5' end of the short DNA chains. A structure for the novel oligonucleotide is proposed to be p5' X3' pp5' rN, where X represents unidentified nucleoside with a peculiar property. Possible metabolic relationship between these molecules and primer RNA has been discussed.     

In vitro polyoma DNA synthesis: Involvement of RNA in discontinuous chain growth

Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-³²P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-³²P-labeled deoxynucleoside triphosphates showed ³²P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived.     

Transcriptional role in DNA replication: degradation of RNA primer during DNA synthesis

DNA synthesis catalyzed in vitro by E. coli DNA polymeraseI in the presence of single stranded fd DNA or poly (dT) as template is stimulated by RNA primers. When poly(dT) fully or partially saturated with polyriboadenylic acid strands is used as template - primer, DNA synthesis proceeds with concomitant degradation of the ribostrands to 5′-adenosine monophosphate. The fragment of DNA polymerase lacking the 5′→3′ exonuclease shows comparable RNA primer dependency but reduced efficiency for the degradation of the RNA primer from the 5′-end.     

Regulation of the in vitro synthesis of the alpha-peptide of beta-galactosidase directed by a restriction fragment of the lactose operon.

The regulation of the $\text{in vitro}$ synthesis of the N-terminal portion of the β-galactosidase molecule (α-peptide) has been investigated using DNA fragments of the lactose operon as template. DNA fragments of about 789 base pairs were isolated after endonuclease (Hin II) digestion of either λplac5, λh80dlacp^s or λh80dlacUV5 phage DNA or DNA from the recombinant plasmid PMC3. The regulation of the expression of these fragments is similar to that observed for the synthesis of β-galactosidase using total phage or plasmid DNA as template, indicating that the regulatory regions on the fragments are intact and functional. Thus, the synthesis of the α-peptide required an inducer due to the presence of $\text{lac}$ repressor in the $\text{E. coli}$ S-30 extract used. In addition a dependency on adenosine 3′,5′-cyclic monophosphate (cAMP)¹ for α-peptide synthesis was obtained with the fragments isolated from λplac5 and λh80dlacp^s DNAs, whereas little effect of cAMP was seen with the fragment isolated from λh80dlacUV5 phage DNA or PMC3 plasmid DNA containing a UV5 promotor region. However, a significant difference in the effect of guanosine-3′-diphosphate-5′-diphosphate (ppGpp) was observed. With the total phage DNA as template, ppGpp resulted in a 2–4 fold stimulation whereas with the fragment, or PMC3 plasmid DNA, directed synthesis of the α-peptide no significant stimulation by ppGpp was seen.