Initiation of RNA-primed DNA synthesis<i>in vitro</i>by DNA polymerase α-primase

Harrington, Cindy; Perrino, Fred W.

doi:10.1093/nar/23.6.1003

Cited by 15 publications

(19 citation statements)

References 37 publications

(28 reference statements)

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“…DNA pol ␣/primase acts by first adding the eventual second nucleotide of the RNA sequence, preferring this nucleotide to be a purine. More recently, a preferential priming sequence 5Ј-d(GCTTTCTTCC) has been deduced in vitro (20).…”

Section: Resultsmentioning

confidence: 99%

Synthesis of the Mammalian Telomere Lagging Strand in Vitro

Reveal

Henkels

Turchi

1997

Journal of Biological Chemistry

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Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy-and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase ␣. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase ␣/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase ␣ on the d(T-TAGGG) 10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3 thymidine nucleotide generating a five-base RNA primer with the sequence 5-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase ␣. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.Semiconservative discontinuous bidirectional DNA replication presents the "end problem" for replication of linear genomes (1). Mammalian chromosomes contain short repeated DNA sequences complexed with specific proteins at each termini (telomeres) and, in conjunction with telomerase, can circumvent this problem. Telomerase has been extensively studied and catalyzes the 5Ј to 3Ј extension of the terminal 3Ј-OH using an internal ribonucleotide template (reviewed in Ref. 2). To completely replicate telomeres, the strand complementary to the telomeric leading strand must be synthesized to generate a double strand DNA product.The mechanism by which the DNA strand complementary to the telomerase catalyzed leading strand is synthesized has not been addressed experimentally. Two models have been proposed for synthesis of the telomere lagging strand. The first involves RNA priming and DNA extension followed by removal of RNA primers and ligation of the Okazaki-like fragments (3, 4), presumably using the same enzymatic machinery employed for lagging strand synthesis at a replication fork (5, 6). The alternative hypothesis is based upon in vitro analyses of telomeric DNA sequences and the propensity to form higher ordered structures (7). The structure generated must present a 3Ј-hydroxyl for DNA polymerase (pol) 1 catalyzed extension for synthesis of the lagging strand followed by a nucleolytic processing reaction to generate a terminal 3Ј-hydroxyl to enable telomerase extension in the next round of replication. Experimental evidence for either of these hypoth...

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Section: Resultsmentioning

confidence: 99%

Synthesis of the Mammalian Telomere Lagging Strand in Vitro

Reveal

Henkels

Turchi

1997

Journal of Biological Chemistry

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“…Primase initiates synthesis by insertion of the first 5Ј-GTP at nucleotide 31. Nucleotide 23 is seven nucleotides from the initiation start site (16). These two positions were selected to test for differences in primer synthesis that might relate to the position of araCMP within the priming site.…”

Section: Insertion Of Aracmp Into Full-lengthmentioning

confidence: 99%

“…Primer initiation sites are rich in thymidine and cytosine residues (13,15,16,26). The high level of thymidine residues should make it more likely that FaraAMP and araAMP might be incorporated into elongating primers by primase within the first 6 bases, resulting in truncated primers that are not elongated by pol ␣.…”

Section: Insertion Of Aracmp Into Full-lengthmentioning

confidence: 99%

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The Effects of Cytosine Arabinoside on RNA-primed DNA Synthesis by DNA Polymerase α-Primase

Harrington

Perrino

1995

Journal of Biological Chemistry

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Oligonucleotides containing a specific initiation site for polymerase ␣-primase (pol ␣-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3 terminus of a full-length RNA primer with a 400-fold preference over CMP. The araCMP is elongated efficiently by pol ␣ in the primasecoupled reaction. Extension from RNA 3-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol ␣ is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3 terminus. These results show that the pol ␣-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol ␣ and less so from inhibition of primer synthesis by primase.In cells, RNA-primed DNA fragments are synthesized at origins of replication and on lagging strands at replication forks (1, 2). Initiation of DNA chains requires oligoribonucleotide synthesis to provide 3Ј-hydroxyl termini for DNA polymerase elongation. The tight association between DNA polymerase ␣ (pol ␣) 1 and primase indicate that pol ␣-primase complex is responsible for RNA-primed DNA synthesis. Primase synthesizes RNA primers that are about 10 Nts in length de novo, and pol ␣ elongates the primers.The aranucleotides inhibit DNA replication in animal cells (3). Inhibition by the triphosphate forms of these analogues might result during RNA-primed DNA synthesis by pol ␣-primase (4). Incorporation into RNA primers by primase and into DNA by pol ␣ is at positions opposite complementary nucleotides (3-10). Primase inserts araATP and FaraATP more efficiently than ATP (9, 11), and pol ␣ inserts these analogues as efficiently as deoxynucleotides (12). Insertion of aranucleotides terminates primer chain elongation by primase (9, 11), and 3Ј-aranucleotides in DNA decrease elongation by pol ␣ 2000-fold (12). In contrast, aranucleotides at RNA 3Ј termini only moderately decrease elongation by pol ␣ (9, 11). The synthesis of nonfunctional primers less than 7 Nts in length using homopolymer templates has made it difficult to quantitate effects of aranucleotides during RNA-primed DNA synthesis and to distinguish effects on primase and pol ␣.Oligonucleotide templates permit analysis of sequence-specific effects of aranucleotides on RNA-primed DNA synthesis. The sequences of some primer initiation sites have been identified (13-16), and these sequences support initiation in oligomer templates (15, 16). Using an oligonucleotide containing an initiation site for pol ␣-primase, primers with the sequence 5Ј-GGAAGAAAGC-3Ј are generated (16). Incorporation of araCMP at the 3Ј terminus...

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“…We suspect that this result may be related to the extent of unwinding that takes place in the two reactions. For instance, it has been reported that the priming sites nearest a given replication fork are preferentially used (36). Walter and Newport discuss additional evidence that the DNA Pol ␣-primase complex specifically binds to forks formed during DNA unwinding (80).…”

Section: Vol 75 2001 Replication Initiation Site Selection In Vitromentioning

confidence: 99%

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

Purviance

Prack

Barbaro

et al. 2001

J Virol

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The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) ␣-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol ␣-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.Studies employing the simian virus 40 (SV40) in vitro replication system have been used to establish much of what is known about the enzymology of eukaryotic DNA replication (10,40,81). This system has also been used to study the reaction mechanisms that take place during particular stages in the replication process; for instance, during the initiation of DNA replication. SV40 replication is initiated when T antigen (T-ag), the single viral protein necessary for replication, site specifically binds to the viral origin (reviewed in references 8, 11, and 31). Upon binding, T-ag assembles into a double hexamer (21,23,51,61,77) that is able to function as a helicase (22,34,66,68,84). Owing to its helicase activity, T-ag is capable of catalyzing origin-specific unwinding, provided replication protein A (RPA) and topoisomerase I are present in the reaction (15,22,27,88).Recent insights into initiation of DNA replication at the SV40 origin include images of T-ag double hexamers assembled on the viral core origin (77). Additional studies have helped to define the minimal core origin sequences necessary for T-ag double hexamer formation (41,42,67). Further insights into T-ag's interactions with the central region of the core origin, site II, were provided by the solution structure of the T-ag origin binding domain (T-ag-obd 131-260 ) (49). These studies established that a pair of loops is utilized to make sequence-specific contacts with site II (49); a similar surface is used by the DNA-binding domain of papillomavirus to bind to DNA (29). Recent experiments have also helped to unravel how T-ag's interactions with the core origin are controlled by the cell cycle machinery (5; reference 83 and references therein).The SV40 replication system has also been used to investigate the formation of nascent DNA in the vicinity of the SV40 origin following T-ag cataly...

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Initiation of RNA-primed DNA synthesisin vitroby DNA polymerase α-primase

Cited by 15 publications

References 37 publications

Synthesis of the Mammalian Telomere Lagging Strand in Vitro

Synthesis of the Mammalian Telomere Lagging Strand in Vitro

The Effects of Cytosine Arabinoside on RNA-primed DNA Synthesis by DNA Polymerase α-Primase

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

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