Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

Replication of polyoma DNA in isolated nuclei. VII. Initiator RNA synthesis during nucleotide depletion.

Earlier experiments demonstrated that the Okazaki fragments synthesized during discontinuous polyoma DNA synthesis in isolated nuclei at their 5′ ends contained structural elements consisting of polyribonucleotides starting with ATP or GTP (Reichard et al., 1974). These structures could be released by digestion with pancreatic DNAase and were named initiator RNA. They consist of a large family of polyribonucleotides differing in base sequence but having a common size of about a decanucleotide. We now demonstrate that limitation of DNA synthesis by low concentrations of deoxyribonucleoside triphosphates in parallel limits the synthesis of initiator RNA. This is additional evidence for the primer function of initiator RNA. When ribonucleoside triphosphates other than ATP were deleted from the incubation medium only a small decrease of DNA and initiator RNA synthesis occurred. Under those conditions deoxyribonucleotides substituted for ribonucleotides and were incorporated internally into the primer. From this result as well as the insensitivity of initiator RNA synthesis to α-amanitin (Reichard &; Eliasson, 1979) we suggest that a mammalian counterpart to primase, the dnaG gene product of Escherichia coli(Rowen &; Kornberg, 1978a), catalyzes the synthesis of initiator RNA.     

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.     

Origin and degradation of the RNA primers at the 5' termini of nascent DNA chains in Bacillus subtilis

We had earlier characterized the nascent DNA synthesized in permeable cells of Bacillus subtilis in the presence of 5-mercurideoxycytidine triphosphate and 2',3'-dideoxyATP as being substituted at its 5' end with a ribonucleotide moiety of the sequence pApG(pC)1-2 DNA. In this paper, we examine the origin and turnover of the DNA-linked ribonucleotide and its relationship to DNA replication. At least 50% of the RNA-linked nascent DNA chains served as guanylate acceptors when incubated with GTP and the eukaryotic capping enzyme, indicating the presence of 5'-terminal di- or triphosphate groups and suggesting that the RNA moiety is synthesized de novo and is not a degradation product. In nascent DNA produced without limitation of chain growth by dideoxyATP, the degree of terminal ribonucleotide substitution was reduced by 50%, consistent with a linkage between RNA primer removal and DNA chain growth. Such a relationship was demonstrated directly by examining the RNA primer content of nascent DNA synthesized in the absence of dideoxyATP as a function of DNA chain length. As the DNA size increased from 40 to 200 nucleotide residues, the extent of RNA substitution declined from 80% to nearly 0%. Endgroup analysis showed that the loss of RNA was accompanied by a gradual shift from predominantly adenylate residues to 5'-terminal guanylate, consistent with a stepwise removal of ribonucleotides from the 5' end. Evidence that the nascent mercurated DNA synthesized under our experimental conditions was indeed a replicative intermediate came from the study of the time course of DNA chain growth and pulse-chase experiments. In the presence of the DNA ligase inhibitor NMN, mercurated DNA accumulated in two size classes with average length of approximately 750 and 8000 nucleotide residues, presumably representing the mature size of intermediates in discontinuous DNA synthesis. Comparison with the DNA size range at which the loss of the 5'-terminal RNA moiety occurred (40 to 200 residues) indicated that the processing of RNA primers occurred at an early stage during DNA chain elongation, and that moderate size intermediates in discontinuous DNA replication (greater than 200 nucleotides) have already lost their RNA primers.     

Differential effect of neomycin on DNA dependent -DNA and RNA synthesis in vitro

Neomycin inhibits $\text{in vitro}$ DNA dependent DNA and RNA synthesis catalyzed by DNA polymerase I and RNA polymerase from $\text{E. coli}$. The effect of the antibiotic is more pronounced towards DNA synthesis. The inhibition of DNA synthesis is competitive with template DNA, does not reverse with excess deoxynucleoside triphosphate, Mg²⁺ or enzyme $\text{E. coli}$ DNA polymerase I. Neomycin does not reduce the number of potential 3′ -OH end or primer. It seems to shorten the size of the newly formed polynucleotide.     

The role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an $\text{E. coli}$ mutant deficient in the 5′ → 3′ exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.     

Replication protein A modulates its interface with the primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes

The eukaryal single-stranded DNA binding protein replication protein A (RPA) binds short oligonucleotides with high affinity but exhibits low cooperativity in binding longer templates, opposite to prokaryal counterparts. This discrepancy could reflect the smaller size of the replicative template portion availed to RPA. According to current models, this portion accommodates an RNA–DNA primer (RDP) of <40 nt (nested discontinuity) or a several-fold longer Okazaki fragment (initiation zone). Previous in situ UV-crosslinking revealed that RPA also interacts with nascent DNA, especially growing RDPs. Here we compare nascent SV40 DNA chains UV-crosslinked to the middle and large RPA subunits and use the data to re-examine the two models. The middle subunit interacted with the nascent chains after a few DNA residues were added to the RNA primer while the large subunit became accessible after extension by several more. Upon RDP maturation, the middle subunit disengaged while the large subunit remained accessible during further limited extension. A corresponding shift in preference in favor of the large subunit has been reported for purified RPA and synthetic gapped duplexes upon reduction of the gap from 19 to 9 nt. Combined, these facts support the proposal that the mature RDP faces downstream a correspondingly small gap, possibly created by removal of the RNA primer moiety from an adjacent, previously synthesized RDP (nested discontinuity) but insufficient for continuous elongation of the RDP into an Okazaki fragment (initiation zone).     

Ligation of highly modified bacteriophage DNA

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4′,5′-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, λ DNA to be ligated was as follows: $\text{λ}\text{DNA}\text{=}\text{XP}\text{12}\text{DNA}\text{>}\text{SP}\text{82}\text{DNA}\text{?}\text{nonglucosylated}\text{T}\text{4}\text{DNA}\text{>}\text{T}\text{4}\text{DNA}\text{=}\text{PBS}\text{1}\text{DNA}\text{?}\text{SP}\text{15}\text{DNA}$. TaqI-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and λ DNAs was equally efficient. We conclude that the poor ligation of TaqI fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.     

Initiator RNA of discontinuous DNA synthesis in human lymphocytes

A short RNA covalently associated with nascent DNA has been isolated after synthesis in vitro with labeled ribonucleoside triphosphates and the removal of DNA by DNAase digestion. The RNA migrates in polyacrylamide gels or chromatographs on DEAE-Sephadex columns as a relatively discrete oligonucleotide 8–11 nucleotides in length. The RNA is associated primarily with nascent DNA with stoichiometry of approximately one per DNA chain. The RNA has a triphosphate group at the 5′ end and 2 or 3 deoxynucleotide residues at the 3′ end that are not removed by DNAase. These results further support a role for the RNA as an initiator of discontinuous DNA synthesis. Examination of sequences present at the 3′ end of the RNA using RNAase to effect transfer of ³²PO₄ from ³²P-labeled DNA to covalently attached RNA indicates that a diverse, rather than unique, set of sequences are present in the RNA.     

Transcription of polyoma virus DNA after interaction with nuclear proteins in vitro.

Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent ³²PP_i exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase.     

In vitro complex formation between bacteriophage φ29 terminal protein and deoxynucleotide

It has previously been shown that the 5′-terminal deoxyadenosine residue of each φ29 DNA strand is linked covalently to the 30,000 dalton terminal protein. When extracts prepared from φ29-infected$\text{Bacillus}\text{subtilis}$ cells are incubated with [α-³²p]dATP, complexes consisting of the nucleotide covalently linked to a 30,000 dalton protein can be detected. The formation of this complex requires the presence of φ29 DNA containing the bound 30,000 dalton terminal protein and Mg⁺⁺. When uninfected cell extracts were used, there was no complex formation. When [α-³²p]dCTP was used in place of [α-³²p]dATP, no complex was formed. DNA-protein templates prepared from φ29 related phages, φ15, Nf, M2Y and GA-1, also supported the complex formation in various degrees. These results support the hypothesis that the terminal protein serves as a primer for the initiation of φ29 DNA replication.     

Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells.

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides.Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ-³²P]ATP. After selective degradation of the DNA portions, [5′-³²P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.     

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.     

RNA-directed DNA polymerase of Rous sarcoma virus: initiation of synthesis with 70 S viral RNA as template

DNA synthesis by the RNA-directed DNA polymerase of Rous sarcoma virus with 70 S viral RNA as template initiates by the covalent attachment of dAMP to the 3′ terminal adenosine of an RNA molecule. Initiation continues throughout the course of a 90-minute enzymatic reaction, and chain propagation occurs on most if not all of the dAMP residues attached to primer RNA. The nature of the primer molecules was established in two ways. First, the RNA was tagged by attachment of radioactive mono- and oligodeoxynucleotides. Second, primers were isolated directly from their covalent complexes with nascent DNA. The results of both procedures indicate that DNA synthesis initiates on the 3′ termini of 4 S RNA molecules hydrogen-bonded to 70 S RNA. Purified primer RNA has a nucleotide composition (G + C = 64%) different from that (G + C = 60%) of other 4 S RNAs found hydrogen-bonded to the 70 S RNA of Rous sarcoma virus.     

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.     

Isolation of an androgen acceptor from salt extract of rat prostatic chromatin

The soluble androgen acceptor has been isolated from 0.35 M NaCl extract of rat prostatic chromatin by affinity chromatography on DNA-cellulose. The acceptor activity was assayed by interaction with 5α-dihydrotestosterone-receptor. Native DNA enhances this interaction. Polyacrylamide gel electrophoresis of the acceptor under denaturing conditions reveals a single polypeptide of molecular weight of 14,000. Amino acid analysis shows that the acceptor protein contains a higher content of acidic amino acid residues than basic amino acid residues. In an $\text{in}\text{vitro}$ RNA synthesizing system catalyzed by rat RNA polymerase II, addition of the acceptor stimulates RNA synthesis. Based on incorporation of [γ-³²P]ATP and [γ-³²P]GTP, the stimulation by the acceptor is mainly on the initiation of RNA chains.     

Regulation of the in vitro synthesis of E. coli ribosomal protein L12.

The $\text{in}\text{vitro}$ DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized $\text{in}\text{vitro}$ from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene.