Phosphorylation of the DNA Polymerase  -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Ferrari, Marina G.; Lucchini, Giovanna; Plevani, Paolo; Foiani, Marco

doi:10.1074/jbc.271.15.8661

Cited by 28 publications

(29 citation statements)

References 33 publications

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“…The phosphorylation of pol α/primase has been reported in human and S. cerevisiae cells (38)(39)(40); both human p180 and p70 were phosphorylated predominantly during late G2 and M phases of the cell cycle (38). Yeast p86 (B subunit) is phosphorylated in a manner that depends on the stage of the cell cycle (39), but most phosphorylation occurs late in the cell cycle, suggesting that it might play a role either in coordinating replication and mitosis or in resetting the replication apparatus for the next S phase.…”

Section: Dna Polymerase α/Primase Complexmentioning

confidence: 99%

The Dna Replication Fork in Eukaryotic Cells

Waga

Stillman

1998

Annu. Rev. Biochem.

740

653

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Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity. CONTENTS

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Section: Dna Polymerase α/Primase Complexmentioning

confidence: 99%

The Dna Replication Fork in Eukaryotic Cells

Waga

Stillman

1998

Annu. Rev. Biochem.

740

653

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“…T antigen, RP-A, and DNA polymerase a-primase, that are required for initiation of SV40 DNA replication, are potential substrates of Cyclin-dependent kinases (Cdks; Dutta and Stillman 1992;McVey et al, 1989;Nasheuer et al, 1991Nasheuer et al, , 1992. Since RP-A and DNA polymerase a-primase are key cellular replication proteins, and they are phosphorylated in a cell cycle-dependent manner, it was suggested that their phosphorylation might be important for the regulation of DNA replication (Din et al, 1990;Dutta and Stillman 1992;Ferrari et al, 1996;Foiani et al, 1995;Nasheuer et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of DNA polymerase α-primase by Cyclin A-dependent kinases regulates initiation of DNA replication in vitro

Voitenleitner¹,

Fanning

Nasheuer³

1997

Oncogene

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DNA polymerase a-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase aprimase. The p180 and the p68 subunits of DNA polymerase a-primase were phosphorylated using Cyclin A-, B-and E-dependent kinases. This phosphorylation did not in¯uence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, sitespeci®c initiation of replication on plasmid DNA containing the SV40 origin is aected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no eect and Cyclin E-Cdk2 stimulated the initation reaction. DNA polymerase a-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase a-primase remained fully active in the initiation reaction.

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“…Recently, it was reported that p68 specifically plays an essential role at the initial stage of DNA synthesis, before the hydroxyurea-sensitive step, and that the subunit is phosphorylated and dephosphorylated in a cell cycle-dependent manner (11,12). Furthermore, formation of the p180-p68 subcomplex appears to be a prerequisite for p68 phosphorylation (10). However, the physiological role of p68 phosphorylation has been refractory to investigation.…”

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confidence: 99%

The Second-Largest Subunit of the Mouse DNA Polymerase α-Primase Complex Facilitates Both Production and Nuclear Translocation of the Catalytic Subunit of DNA Polymerase α

Mizuno

Ito

Yokoi

et al. 1998

Molecular and Cellular Biology

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DNA polymerase ␣-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase ␣ is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase ␣ complex.Chromosomal DNA replication in eukaryotes is a highly regulated process that requires a large number of replication factors, including distinct three types of DNA polymerases, ␣, ␦, and ε. Studies on the in vitro replication of simian virus 40 (SV40) DNA have made possible the functional identification of several DNA replication factors, including DNA polymerases (4, 17, 35). To date, DNA polymerase ␣-primase has been considered to provide RNA-DNA primers for the initiation of leading-strand synthesis and Okazaki fragment synthesis on the lagging strand during SV40 DNA replication (36,43,44).Mouse DNA polymerase ␣-primase complex was isolated from FM3A cells as a protein complex consisting of four subunits. The apparent molecular masses of these subunits based on their migration on sodium dodecyl sulfate (SDS) polyacrylamide gels are 180, 68, 54, and 46 kDa (39,40). The two smaller subunits, p46 and p54, can be dissociated from the other subunits by treatment with 50% ethylene glycol (38) and possess DNA primase activity, as demonstrated by the synthesis of unit-length oligoribonucleotides (7, 27). The largest subunit, p180, is the catalytic subunit of DNA polymerase and exhibits intrinsic DNA polymerase activity, as revealed in the baculovirus expression system (6). When the human p180 subunit is expressed alone in insect cells, it displays similarities to the holoenzyme such as an identical K m for the primer-template and deoxynucleoside triphosphate and similarities with respect to sensitivity to inhibitors, thermostability, DNA synthetic processivity, and fidelity (6). In contrast, the function of the second-largest subunit, p68, which is tightly bound to p180, remains unclear. To date, no enzymatic activity has been found for p68, and biochemical a...

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Phosphorylation of the DNA Polymerase -Primase B Subunit Is Dependent on Its Association with the p180 Polypeptide

Cited by 28 publications

References 33 publications

The Dna Replication Fork in Eukaryotic Cells

The Dna Replication Fork in Eukaryotic Cells

Phosphorylation of DNA polymerase α-primase by Cyclin A-dependent kinases regulates initiation of DNA replication in vitro

The Second-Largest Subunit of the Mouse DNA Polymerase α-Primase Complex Facilitates Both Production and Nuclear Translocation of the Catalytic Subunit of DNA Polymerase α

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