Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

Voitenleitner, Christian; Rehfuess, Christoph; Hilmes, Melissa A.; O'Rear, Lynda; Liao, Pao Chi; Gage, Douglas A.; Ott, R.; Nasheuer, Heinz-Peter; Fanning, Ellen

doi:10.1128/mcb.19.1.646

Cited by 76 publications

(107 citation statements)

References 59 publications

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“…Alternatively, because p68 itself binds SV40 TAg (32,34,56), active complex formation may require a particular coordination between the interactions of p180 and p68 with TAg, which might be disturbed when the two subunits are derived from different species. Abundant evidence exists that p68 acts as a regulator of DNA polymerase ␣-primase (36,(55)(56)(57)(58), but the suppression of Mp180 C terminus-induced inhibition of SV40 DNA replication by Mp68 is only partial and is not evident in complexes such as (H671M)MH 2 and M 2 H 2 ( Fig. 6A and Ref.…”

Section: Discussionmentioning

confidence: 99%

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Smith¹,

Steffen²,

Grosse³

et al. 2002

Journal of Biological Chemistry

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Human cell extracts support the replication of SV40 DNA, whereas mouse cell extracts do not. Species specificity is determined at the level of initiation of DNA replication, and it was previously found that this requires the large subunit, p180, of DNA polymerase ␣-primase to be of human origin. Furthermore, a functional interaction between SV40 large T antigen (TAg) and p180 is essential for viral DNA replication. In this study we determined that the N-terminal regions of human p180, which contain the TAg-binding sites, can be replaced with those of murine origin without losing the ability to support SV40 DNA replication in vitro. The same substitutions do not prevent SV40 TAg from stimulating the activity of DNA polymerase ␣-primase on single-stranded DNA in the presence of replication protein A. Furthermore, biophysical studies show that the interactions of human and murine DNA polymerase ␣-primase with SV40 TAg are of a similar magnitude. These studies strongly suggest that requirement of SV40 DNA replication for human DNA polymerase ␣ depends neither on the TAg-binding site being of human origin nor on the strength of the binary interaction between SV40 TAg and DNA polymerase ␣-primase but rather on sequences in the C-terminal region of human p180.Polyomavirus DNA replication has been studied extensively because of the ease with which viruses of this family, which include SV40 and mouse polyoma virus (PyV), 1 can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed investigation of the individual factors that play a role in the replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the host DNA but does contribute one essential trans-acting factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at which it forms a double hexamer (3). In the presence of the single-stranded DNA-binding protein, RPA (replication protein A), and topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the DNA polymerase ␣-primase heterotetramer, which then initiates bidirectional DNA synthesis (4 -11). DNA polymerase ␣-primase initiates DNA replication through the action of its smallest subunit, p48, which synthesizes RNA primers that are subsequently elongated by the large subunit, p180 (12-15). The bulk of DNA synthesis is, however, carried out by a more processive enzyme complex consisting of DNA polymerase ␦ and its processivity factor, PCNA (16 -18). The transition between DNA synthesis by DNA polymerases ␣ and ␦ is mediated by the PCNA loading factor replication factor C (16, 19). Throughout the viral replication cycle, TAg double hexamers are thought to act as the replicative helicase (20). For a more extensive account of the DNA replication process and its participating factors see Refs. 1, 2, and 21-23. SV40 and PyV are very similar with regard to DNA replication; their respectiv...

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Section: Discussionmentioning

confidence: 99%

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Smith¹,

Steffen²,

Grosse³

et al. 2002

Journal of Biological Chemistry

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“…Our results argue for the latter, as cyclin A could be shown to stimulate Pol ␦-dependent DNA synthesis. Furthermore, it was recently reported that Pol ␣-dependent initiation of SV40 DNA replication in vitro was stimulated by cyclin E͞cdk2, but inhibited by cyclin A͞cdk2 (40). Thus, by concomitantly inactivating Pol ␣-associated functions and activating Pol ␦ and͞or some of its cofactors, cyclin A may contribute to the switch from Pol ␣-dependent initiation to Pol ␦-driven strand elongation.…”

Section: Discussionmentioning

confidence: 99%

Cyclin A activates the DNA polymerase δ-dependent elongation machinery in vitro : A parvovirus DNA replication model

Bashir

Hörlein

Rommelaere

et al. 2000

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

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Replication of the single-stranded linear DNA genome of parvovirus minute virus of mice (MVM) starts with complementary strand synthesis from the 3 -terminal snap-back telomere, which serves as a primer for the formation of double-stranded replicative form (RF) DNA. This DNA elongation reaction, designated conversion, is exclusively dependent on cellular factors. In cell extracts, we found that complementary strand synthesis was inhibited by the cyclindependent kinase inhibitor p21 WAF1/CIP1 and rescued by the addition of proliferating cell nuclear antigen, arguing for the involvement of DNA polymerase (Pol) ␦ in the conversion reaction. In vivo time course analyses using synchronized MVM-infected A9 cells allowed initial detection of MVM RF DNA at the G1͞S phase transition, coinciding with the onset of cyclin A expression and cyclin A-associated kinase activity. Under in vitro conditions, formation of RF DNA was efficiently supported by A9 S cell extracts, but only marginally by G 1 cell extracts. Addition of recombinant cyclin A stimulated DNA conversion in G 1 cell extracts, and correlated with a concomitant increase in cyclin A-associated kinase activity. Conversely, a specific antibody neutralizing cyclin A-dependent kinase activity, abolished the capacity of S cell extracts for DNA conversion. We found no evidence for the involvement of cyclin E in the regulation of the conversion reaction. We conclude that cyclin A is necessary for activation of complementary strand synthesis, which we propose as a model reaction to study the cell cycle regulation of the Pol ␦-dependent elongation machinery. E ukaryotic DNA replication is restricted to the S phase of the cell cycle. Cell cycle progression is regulated through the action of cyclin-dependent kinases (cdks) that are sequentially activated on association with different cyclins (1). In vertebrate cells, the transition from G 1 to S phase is controlled by the activities of cyclin E-and cyclin A-dependent kinases (2). There is accumulating evidence for the involvement of cyclin A and cyclin E in the regulation of cellular DNA replication. Cyclin A is localized at nuclear replication foci in mammalian cells (3, 4), and both cyclin A and cdk2 have been found to be associated with replicating DNA in the simian virus 40 (SV40) in vitro replication system (5). Addition of recombinant cyclin A͞cdk2 and cyclin E͞cdk2 to G 1 phase nuclei has been shown to trigger the initiation of DNA replication in a human cell-free system (6). Similarly, the inability of human G 1 cell extracts to replicate SV40 origin-containing DNA in vitro could be overcome by the addition of cyclin A or active cdc2 kinase (7,8). Conversely, immunodepletion of cyclin A from S cell extracts partially inhibited SV40 origin-driven plasmid replication (9), whereas a significant decrease in the ability of Xenopus egg extracts to replicate sperm DNA was observed after depletion of cyclin E or cdk2 (10, 11). However, it has been difficult to identify replication factors that are targets for cdks in vivo a...

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“…10), raises the question whether the initiation of leading and lagging strand replication are controlled by different mechanisms. With the recombinant replication proteins in hand and with the known regulation of leading strand initiation by cyclin-dependent kinases (52,75,76), this question can now be addressed.…”

Section: Figmentioning

confidence: 99%

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

Weißhart¹,

Förster²,

Kremmer³

et al. 2000

Journal of Biological Chemistry

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DNA polymerase ␣-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim 2 ) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim 2 indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2⅐10 ؊8 M and 1.3⅐10 ؊8 M, respectively.In eukaryotic cells the duplication of the genome is a highly accurate and tightly coordinated process (reviewed in Ref. 1). For each reaction a specific set of enzymes and accessory proteins is required to replicate chromosomal DNA (1-4). The first step in leading strand DNA replication is accomplished by the synthesis of oligoribonucleotides, called RNA primers, at the origin of DNA replication. This process is carried out by a special enzyme, DNA primase (5-7). The eukaryotic enzyme consists of two subunits, the catalytic subunit p48 and p58, which serves to stabilize the primase activity (8 -13). In eukaryotic cells these two subunits assemble together with the catalytic DNA polymerase subunit, p180, and the p68 polypeptide into a heterotetrameric DNA polymerase ␣-primase complex (pol-prim) 1 (1-7, 14, 15).The initiation of DNA replication requires the interaction of several proteins in vivo and in vitro (1). The start of DNA synthesis de novo occurs by two independent processes: the singular priming event on the leading strand at the origin and the multiple priming steps for Okazaki fragment synthesis on the lagging strand. Both tasks are carried out by the primase activity (1-3, 14 -17). Two cell-free initiation reactions have served as model systems to investigate these processes, primer synthesis in the cell-free SV40 DNA replication system and primer synthesis on natural single-stranded (ss) DNA templates bound by replication protein A (RPA) (17, 18). Using these cell-free systems, it was shown that the initiation of SV40 DNA replication at the origin is species-specific and requires the activity of the p180 subunit of primate pol-prim (19 -21). In contrast to these results, the initiation of Okazaki fragments during lagging strand DNA synthesis is not species-specific and the human as well as the bovine pol-prim can perform lagging strand initiat...

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Cell Cycle-Dependent Regulation of Human DNA Polymerase α-Primase Activity by Phosphorylation

Cited by 76 publications

References 59 publications

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Species Specificity of Simian Virus 40 DNA Replication in Vitro Requires Multiple Functions of Human DNA Polymerase α

Cyclin A activates the DNA polymerase δ-dependent elongation machinery in vitro : A parvovirus DNA replication model

Protein-Protein Interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen Are Required for Efficient Primer Synthesis in a Cell-free System

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