RNA primers in SV40 DNA replication: identification of transient RNA-DNA covalent linkages in replicating DNA

Anderson, Stephen F.; Kaufman, Gregory P.; Ml, DePamphilis

doi:10.1021/bi00642a009

Cited by 73 publications

(34 citation statements)

References 28 publications

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“…The 16 possible combinations expected for a random sequence distribution of RNA-DNA linkages were found, indicating absence of a site-specific initiation of the 4S DNA fragments. This is in agreement with other studies on RNA priming in eukaryotic cells (16,17). From (31).…”

Section: Properties Of the Reconstructed Nuclear Replicationsupporting

confidence: 93%

“…Despite disruption of the cell structure, isolated nuclei support semiconservative DNA synthesis (11, 12), the discontinuous process of viral and/or cellular DNA replication (13)(14)(15), and the initiation of viral Okazaki pieces by RNA primers (16,17) and their conversion into longer DNA chains (14,18,19). Moreover, Seki and Mueller (20), have reported that the "replicase" activity of nuclei can be partly solubilized by mild salt treatment.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Initiation of HeLa cell DNA synthesis in a subnuclear system.

Brun¹,

Weissbach²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Mammalian cells are known to synthesize DNA in discrete stages, the first of which seems to be the formation of DNA pieces 150-200 nucleotides in length that have a s2o value of about 4 S. We have reconstructed a system derived from HeLa cell nuclei that carries out RNA-primed initiation of the synthesis of small (4S) DNA fragments. This synthesis is resistant to high concentrations of a-amanitin and sensitive to antibody directed against RNA polymerase I, suggesting that this enzyme may be involved in the initiation step. The formation of small DNA fragments in this system also requires DNA polymerase a, heat-labile nuclear factor(s), and at least one other nuclear protein.Systems used to study the mechanisms of DNA replication of eukaryotic cells include permeabilized cells (1, 2), cell lysates (3-6), and isolated nuclei (7-10). Despite disruption of the cell structure, isolated nuclei support semiconservative DNA synthesis (11, 12), the discontinuous process of viral and/or cellular DNA replication (13)(14)(15), and the initiation of viral Okazaki pieces by RNA primers (16,17) and their conversion into longer DNA chains (14,18,19). Moreover, Seki and Mueller (20), have reported that the "replicase" activity of nuclei can be partly solubilized by mild salt treatment.In this report we describe optimal conditions for solubilization of the DNA replicase activity and for reconstruction of the DNA synthetic activity of isolated nuclei from HeLa cells. Using such a reconstructed nuclear system, we have found conditions in which DNA synthesis is dependent upon the addition of rNTPs. We present evidence for the involvement of rNTPs in the priming event for DNA synthesis and can now identify at least four components in the initiation step of mammalian DNA synthesis. These components include DNA polymerase a, a RNA polymerase activity with the properties of RNA polymerase I, a heat-labile factor(s) tightly bound to nuclei, and a fourth component solubilized by salt extraction of nuclei. MATERIALS AND METHODSNuclei Preparation. HeLa S3 cells were synchronized by the double thymidine block previously described (21) and collected in mid S phase 5 hr after release of the second thymidine block. Nuclei were prepared from these cells according to the procedure described by Krokan et al. (8), except that Hepes buffer, pH 7.5, was used in place of Tris-HCI, pH 7.5. The detergenttreated nuclei were thoroughly washed in isotonic buffer, pelleted, and stored in 200 X 106 nuclei aliquots in liquid nitrogen.Nuclear Salt Treatment. Nuclei (200 X 106 in 0.5 ml) were suspended in 3 vol of buffer A [10 mM Hepes, pH 7.5/3 mM MgCI2/2 mM ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/1 mM dithioerythritol] containing 0.32 M NaCI and gently stirred for 10 min. After centrifugation at 1000 X g for 10 min, they were resuspended in 5-8 vol of buffer A containing 10% (vol/vol) glycerol and 0.27 M NaCl and stirred for an additional hr at 0-4°. This operation was repeated once with a 30-min stirring period. The...

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Section: Properties Of the Reconstructed Nuclear Replicationsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Initiation of HeLa cell DNA synthesis in a subnuclear system.

Brun¹,

Weissbach²

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…Perhaps the sequence context around a junction determines how much the monoribonucleotide disrupts the helix, which in turn affects substrate recognition and activity of the enzyme. RNA-DNA junctions occur at frequencies that suggest near random distribution on the genome (48,49). This suggests that junction monoribonucleotides will be embedded with a wide range of efficiencies.…”

Section: Discussionmentioning

confidence: 99%

Creation and Removal of Embedded Ribonucleotides in Chromosomal DNA during Mammalian Okazaki Fragment Processing

Rumbaugh

Murante

Shi

et al. 1997

Journal of Biological Chemistry

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Mammalian RNase HI has been shown to specifically cleave the initiator RNA of Okazaki fragments at the RNA-DNA junction, leaving a single ribonucleotide attached to the 5-end of the downstream DNA segment. This monoribonucleotide can then be removed by the mammalian 5-to 3-exo-/endonuclease, a RAD2 homolog-1 (RTH-1) class nuclease, also known as flap endonuclease-1 (FEN-1). Although FEN-1/RTH-1 nuclease often requires an upstream primer for efficient activity, the presence of an upstream primer is usually inhibitory or neutral for removal of this 5-monoribonucleotide. Using model Okazaki fragment substrates, we found that DNA ligase I can seal a 5-monoribonucleotide into DNA. When both ligase and FEN-1/RTH-1 were present simultaneously, some of the 5-monoribonucleotides were ligated into DNA, while others were released. Thus, a 5-monoribonucleotide, particularly one that is made resistant to FEN-1/RTH-1-directed cleavage by extension of an inhibitory upstream primer, can be ligated into the chromosome, despite the presence of FEN-1/RTH-1 nuclease. DNA ligase I was able to seal different monoribonucleotides into the DNA for all substrates tested, with an efficiency of 1-13% that of ligating DNA. These embedded monoribonucleotides can be removed by the combined action of RNase HI, cutting on the 5-side, and FEN-1/RTH-1 nuclease, cleaving on the 3-side. After FEN-1/RTH-1 action and extension by polymerization, DNA ligase I can join the entirely DNA strands to complete repair.During cellular DNA replication, the leading strand is synthesized continuously in the direction of replication fork propagation. An antiparallel template is used for synthesis of the lagging strand, which therefore must be made as a series of discontinuous segments called Okazaki fragments. As the replication fork opens, new upstream fragments are initiated. Each fragment must be independently primed with initiator RNA, which is later removed, prior to joining of the segments into one continuous strand (1). In eukaryotes, initiator RNA removal is achieved, as reviewed in Bambara et al. (2), by the combined action of two nucleases, RNase HI and a 5Ј-to 3Ј-exo-/endonuclease, called RTH-1 or FEN-1 (3-7). RNase HI makes a structure-specific cleavage, releasing the initiator RNA as an intact segment but leaving a single ribonucleotide on the 5Ј-end of the downstream DNA. FEN-1/RTH-1 nuclease can then remove this monoribonucleotide (8). Although FEN-1/RTH-1 cleavage often requires an upstream primer for stimulation, the presence of an upstream primer is sometimes neutral or even inhibitory, especially for removal of these monoribonucleotides (9). If an upstream primer approaches before FEN-1/RTH-1 action and inhibition at the particular junction is significant, Okazaki fragment processing may be halted, leaving a nick in the chromosome, just upstream of a single ribonucleotide. In such a situation, processing might still be completed via the endonucleolytic activity of FEN-1/RTH-1 nuclease (9). However, if DNA ligase is able to seal such monoribonuc...

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“…Heat-labile and heat-stabile protein factors that stimulate endogenous DNA synthesis can be leached out of nuclei (1,2,(164)(165)(166)(167)(168)(169)(170)(171)(172)(173)(174)(175)(176)(177)(178)295); however, none have been sufficiently characterized to evaluate their significance. In general, they act by extending the period of DNA synthesis and by stimulat ing the joining of Okazaki fragments.…”

Section: Miscellaneous Proteinsmentioning

confidence: 99%

“…Purified replicating SV40 DNA, briefl y labeled in intact cells (183)(184)(185) or subcellular systems (69, 72, 74, 75, 168, 169, 186-188, 190, 19 1) contain 50% or less of their nascent DNA in the form of short chains with an average length of 135 nucleotides (168,192), even when measured after very short labeling periods (169,184,187) or by labeling 5' ends (190). Continued incubation results in a rapid, quantitative joining of Okazaki fragments to longer nascent DNA chains.…”

Section: Semidiscontinuous Dna Synthesismentioning

confidence: 99%