RNA-DNA Junction Research Articles

Yeast ribonuclease H(70) cleaves RNA-DNA junctions

A specific substrate, M 13 DNA:RNA-[32P]DNA, was synthesized to investigate the mode of cleavage of enzymes with RNase H activity. RNase H(70) from Saccharomyces cerevisiae hydrolyzes the phosphodiester bond at the RNA-DNA junction of this substrate, thereby producing a 5'-monophosphate-terminated polydeoxyribonucleotide and 3'-hydroxyl-terminated oligoribonucleotides.

Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Analysis of the nucleotide sequences at the 5′ ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3′-purine-dT-5′ in the DNA template and the secondary site was 3′-purine-dC-5′, with the 5′ end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3′-purine-dT-5′ sites and 20% of the 3′-purine-dC-5′ sites were used. Purine-rich sites, such as PuPuPu and PyPuPu, were excluded. The 5′-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5′ end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region ( DePamphilis et al., 1980 DePamphilis et al., 1983 Hay and DePamphilis, 1982Hay & DePamphilis, 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.

Initiation site of deoxyribonucleotide polymerization at the replication origin of the Escherichia coli chromosome.

A new round of chromosomal replication of a temperature-sensitive initiation mutant (dnaC) of Escherichia coli was initiated synchronously by a temperature shift from a nonpermissive to a permissive condition in the presence of arabinosyl cytosine. Increased amounts of nascent DNA fragments with homology for the chromosomal segment containing the replication origin (oriC) were found. The nascent DNA fragments were purified and treated with alkali to hydrolyze putative primer RNA and to expose 5'-hydroxyl DNA ends at the RNA-DNA junctions. The ends were then labeled selectively with T4 polynucleotide kinase and [gamma-32P]ATP at 0 degrees C and the terminally-labeled initiation fragments were purified by hybridization with origin probe DNAs containing one each of the constituent strands of oriC-DNA segment. The 32P-labeled initiation sites were then located at the resolution of single nucleotides in the nucleotide sequence of the oriC segment after cleavage with restriction enzymes. Two initiation sites of DNA synthesis, 37 nucleotides apart, were detected in one of the component strands of the oriC; in other words, in the strand whose 5' to 3' polynucleotide polarity lies counterclockwise on the E. coli genetic map. The results support the involvement of the primer RNA in the initiation of DNA synthesis at the origin of the E. coli genome and suggest that the first initiation event is asymmetric.

Flexibility in RNA priming of Okazaki pieces at the E. coli replication fork.

We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E. coli replication fork. Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces. All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used. However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction. A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture. Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available.

RNA involvement in T4 DNA synthesis in toluene-treated cells

In T4-infected cells made permeable with toluene, pulses with [(alpha-32P deoxyribonucleoside triphosphates demonstrated covalent linkage of RNA to DNA of the Okazaki fragments. Analysis of the transfer of the 32P label to the 2'(3') ribonucleoside monophosphates indicated that the 3'-end of the RNA primer is heterogeneous. The most frequently encountered ribonucleotide was rCMP, but also transfer to rUMP, rAMP and rGMP occurred at different frequencies. In contrast, no heterogeneity was observed for the deoxyribonucleoside at the RNA-DNA junction. Of all the [to-32P] deoxyribonucleoside triphosphates tested, transfer of the 32P label to 2'(3') rNMPs was predominant when [alpha32P] dGTP was the substrate, indicating that the deoxyribonucleoside most frequently encountered at the RNA-DNA linkage is dG. These observations suggest that the starts for the Okazaki fragments may occur at unique sites of the T4 genome.

Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.

Ribonucleotides in DNA newly synthesised in 3T6 cells in vivo.

Within the field of DNA replication, considerable interest has focused in recent years on the mechanism of initiation of synthesis of DNA molecules. In vitro replication systems from Escherichia coli have been instrumental in uncovering a priming function fo9r ribonucleotides on the earliest intermediates of DNA polymerisation in vitro and in identifying the proteins involved. In vitro replication systems from mammalian cells that permit the use of the phosphate-transfer method for detection of RNA-DNA junctions as well as direct labelling of the RNA moiety of the molecules have suggested a similar role for ribonucleotides in DNA synthesis in eukaryotes. However, the existence of this mechanism in mammalian cells in vivo has not been established. Here we report the first evidence that a significant proportion of the earliest intermediates in mammalian DNA polymerisation in vivo do, in fact, possess ribonucleotides, presumably because their synthesis was initiated with one or more ribonucleotides.

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 T 2 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 T 2 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.

Evidence for covalent association of RNA with nascent DNA in human lymphocytes.

An in vitro DNA synthesizing system prepared from lysates of a human lymphocyte cell line was employed to test for the existence of RNA-DNA covalent bonds. The procedure utilized a form of nearest-neighbor analysis in which 32P was transferred from incorporated α-32P-labeled deoxyribonucleotides in DNA to ribonucleotides after alkaline hydrolysis. All ribonucleotides and deoxyribonucleotides are found in approximately equivalent proportions at the RNA-DNA junction. The RNA-DNA bonds are found primarily at the same density as DNA; they are located at or near the termini of chains; the strands containing the RNA-DNA bonds are predominantly the size of nascent DNA; there is one such bond per DNA chain; and the RNA-DNA bonds are labile during DNA synthesis although stabilized by an inhibitor of synthesis. These properties are consistent with the role of an RNA for the priming of nascent DNA synthesis.

Covalent linkage between RNA and nascent DNA in the slime mold, Physarum polycephalum

When α- 32P-labeled deoxyribonucleoside triphosphates are injected into plasmodia of the eukaryotic slime mold, Physarum polycephalum, they are incorporated initially into strands of DNA which are mostly less than 300 nucleotides long. Sixty minutes after injection incorporated deoxyribonucleoside triphosphates are found in much longer strands. If the short strands found two minutes after injection are denatured and centrifuged to equilibrium in a Cs 2SO 4 density gradient, they migrate to a density slightly greater than that of single-stranded Physarum DNA. When these short strands are treated with alkali to hydrolyze RNA, a small fraction of the incorporated 32P is made acid-soluble and is identified as a mixture of the four ribonucleoside 2′,3′-monophosphates. Such transfer of 32P to ribonucleotides occurs when any of the 4 α- 32P-labeled deoxyribonucleoside triphosphates is used for injection, but the transfer is greatest with [α- 32P]dGTP. We conclude that very short stretches of RNA are found linked through phosphodiester bonds to nascent DNA chains in Physarum polycephalum and that any of the 16 possible combinations of ribo-and deoxyribonucleotides can occur at the RNA-DNA junction.

Replication of polyoma DNA in isolated nuclei: III. The nucleotide sequence at the RNA—DNA junction of nascent strands

Short fragments consisting of about 100 to 140 deoxyribonucleotides serve as intermediates in the elongation of polyoma DNA. In nuclei isolated from polyoma-infected 3T6 mouse fibroblasts these fragments are initiated by stretches of RNA. We investigated the nature of the ribo- and deoxyribonucleotides at the RNA-DNA link. DNA was synthesized in vitro from each of the four α- 32P-labelled deoxynucleoside triphosphates, the nascent strands were hydrolysed with alkali and the transfer of isotope to ribonucleotides was studied after fractionation of strands according to size. Each strand contained on the average one RNA-DNA link at the 5′ end of DNA. All four common ribo- and deoxyribonucleotides were present at the RNA-DNA link with close to equal frequency, irrespective of chain length or incubation time. In a second approach, daughter strands synthesized in vivo were treated with alkali and the 5′-OH ends of DNA liberated were 32P-labelled using polynucleotide kinase. All four deoxynucleotides were labelled by this treatment confirming the corresponding results of the in vitro experiments. During the discontinuous synthesis of polyoma DNA the switch from RNA to DNA synthesis is thus not effected by a specific sequence at the RNA-DNA junction, in contrast to Escherichia coli where the sequence p(rPy)p(dC)p was reported.

Synthesis of herpes simplex virus type 1 (HSV-1) DNA in isolated nuclei. II. Covalent linkage of RNA to nascent viral DNA.

Nascent HSV-1 DNA synthesized in vitro by isolated nuclei of infected rat embryo fibroblasts was found to be covalently linked to stretches of ribonucleotides. The nature of ribo- and deoxyribonucleotides at the RNA-DNA joint was investigated by assaying for the transfer of [32P]-radioisotope from an [α-32P]-deoxyribonucleotide to a 2’(3’)-ribonucleotide after alkali hydrolysis of the nascent HSV-1 DNA. All four common ribo-and deoxyribonucleotides were present at the RNA-DNA junction. To provide additional proof that the RNA pieces were linked to the newly synthesized DNA by a phosphodiester bond, the viral DNA was synthesized in the presence of labeled substrates for both DNA (α-32P-dCTP) and RNA (14C-GTP and14C-CTP). Analysis of the native and heat-denatured viral DNA-RNA copolymers by equilibrium centrifugation in CS2SO4 density gradients indicated that the [14C]-RNA did not dissociate upon denaturation and banded with the [³²P]-DNA.

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 …

RNA-linked DNA fragments in vitro.

RNA-linked DNA fragments are intermediates in DNA replication in Escherichia coli cells made permeable to nucleoside triphosphates by treatment with toluene. Covalent linkage of a short RNA stretch to the 5' end of the DNA is proved by transfer of (32)P from [alpha-(32)P]dNTP to ribonucleotides upon digestion with alkali or pancreatic RNase, and by a small decrease in the molecular size upon alkaline hydrolysis. The (32)P transfer experiments reveal a unique structure...p(rPy)p(rA)p(rU or rC)p(dC)p... at the RNA-DNA junction.

Nucleotides at the RNA-DNA covalent bonds formed in the endogenous reaction by the avian myeloblastosis virus DNA polymerase

Avian myeloblastosis virus (AMV) DNA polymerase initiates in vitro DNA synthesis by a specific covalent joining of the new DNA strands onto tumor virus RNAs. Only certain deoxy- and ribonucleotides are found at the RNADNA covalent bond(s). Only dCMP is joined to the 3′-hydroxyl of rUMP and only dAMP is joined to rAMP. Thus, at least two DNA molecules are formed in the AMV endogenous reaction. dTMP and dGMP apparently do not serve as the first deoxynucleotides at the RNADNA junction. The results are discussed with respect to the termini and structure of the viral RNAs.