RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

Suzuki, Motoshi; Savoysky, Ericka; Izuta, Shunji; Tatebe, Masahiro; Okajima, Tetsuya; Yoshida, Shonen

doi:10.1021/bi00210a030

Cited by 26 publications

(41 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is possible that during the T-ag-induced melting of double-stranded telomeric DNA in the replication reaction, the G-rich strand forms a G-quartet structure, which does not serve as an efficient template for the DNA replication. In addition, it is known that mammalian DNA polymerase ␣-primase preferentially initiates priming at or near pyrimidine-rich (more than six consecutive pyrimidines) sequences (9,35). This is clearly not the case in mammalian telomeric G-rich strands since they have only two thymines in one repeat.…”

Section: Discussionmentioning

confidence: 99%

In Vitro Reconstitution of the End Replication Problem

Ohki

Tsurimoto

Ishikawa

2001

Molecular and Cellular Biology

View full text Add to dashboard Cite

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal ϳ500-bp region, leaving 3 overhangs. This result is consistent with observations in telomerasenegative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.Semiconservative DNA replication is achieved by a harmonious cooperation between two distinct modes of DNA synthesis, leading and lagging strand DNA syntheses. These processes have been extensively characterized in vitro using the simian virus 40 (SV40) DNA replication system, the results of which are summarized as follows (reviewed in reference 37). In leading strand synthesis, the direction of DNA synthesis is the same as that of the replication fork movement. Consequently, leading strand synthesis, performed by DNA polymerase ␦ and ε, is processive. On the other hand, in lagging strand synthesis, DNA is synthesized in a direction opposite to replication fork movement, as short pieces that are eventually ligated to form a continuous DNA strand. This process involves repeated synthesis of RNA primers (ϳ10 nucleotides [nt]), which are elongated into Okazaki fragments. The RNA primer synthesis and the initial phase of DNA elongation (up to 40 nt) are carried out by the DNA polymerase ␣-primase complex, which produces RNA-DNA primers. Subsequently, a DNA polymerase switch takes place, and the RNA-DNA primer is elongated by DNA polymerase ␦ to the full length of its respective Okazaki fragment. Finally, consecutive Okazaki fragments are ligated by removal of RNA primers to form an uninterrupted progeny strand.In replication of linear DNA molecules, the 3Ј end of the nascent strand is synthesized by leading strand synthesis, whereas the 5Ј end is synthesized by lagging strand synthesis. In the early 1970s, it was first suggested that the lagging strand synthesis of linear DNA templates would be incomplete for two reasons (28, 39). First, there is no known mechanism that ensures priming of the most distal Okazaki fragment synthesis from the very end of the template molecule. Accordingly, the template sequence b...

show abstract

Section: Discussionmentioning

confidence: 99%

In Vitro Reconstitution of the End Replication Problem

Ohki

Tsurimoto

Ishikawa

2001

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…Related studies demonstrated that the preferred homopolymer substrate for human (21) and mouse (68) pol ot-primase is poly(dT). Several studies have suggested that DNA primase prefers to start at either deoxythymidine-rich sequences (25,74,75) or pyrimidine clusters (59). Collectively, these studies indicated that pyrimidines, particularly thymines, play a critical role in determining where the pol a-primase complex initiates DNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Bullock¹,

Tevosian²,

Jones³

et al. 1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

Primer RNA-DNA, a small (-30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase a-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.DNA replication in higher eukaryotes is a poorly understood process. This is due, in part, to the failure to establish in vitro replication systems that duplicate DNA molecules from higher eukaryotic organisms. Therefore, important features of eukaryotic DNA replication, such as the enzymology and basic mechanisms that operate during synthesis of DNA, are being established by using in vitro replication systems that are dependent on viral origins.The small papovavirus simian virus 40 (SV40) has served as a useful model for studies of eukaryotic replication. This virus has both a well-defined origin of replication (reference 12 and references therein) and, with the exception of one viral protein termed T antigen (T-ag), utilizes host proteins to synthesize viral DNA (60). Furthermore, an SV40 in vitro replication system that depends on proteins from cells permissive for viral replication (either simian or human) has been developed (38,58,69). This system has enabled the identity and functions of many of the proteins required to replicate SV40 DNA to be established (for reviews, see references 7, 29, and 57).Initiation of DNA replication, including the initiation of SV40 DNA replication, has been the focus of many recent studies. Initiation of SV40 DNA replication has been subdivided into presynthesis and postsynthesis events. Much of what is known about the presynthesis events has been recently reviewed (2,19). In summary, T-ag assembles as a double hexamer at the SV40 origin (40) and melts the early palindrome (3). The inherent helicase activity of T-ag (56) Less is known about events that take place immediately after the initiation of DNA synthesis. We have been using pulse and pulse-chase techniques to investigate the mechanism of initiation of DNA s...

show abstract

“…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%

“…The sequences of some primer initiation sites have been identified (13)(14)(15)(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).…”

mentioning

confidence: 99%

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

Harrington

Perrino

1995

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

Cited by 26 publications

References 27 publications

In Vitro Reconstitution of the End Replication Problem

In Vitro Reconstitution of the End Replication Problem

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

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

Contact Info

Product

Resources

About