Primase Activity of Human DNA Polymerase α-Primase

Schneider, A; Smith, Richard; Kautz, Armin R.; Weißhart, Klaus; Grosse, Frank; Nasheuer, Heinz-Peter

doi:10.1074/jbc.273.34.21608

Cited by 48 publications

(39 citation statements)

References 47 publications

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“…1, lane 7). The purified cloned pol ␣-primase complex contained polymerase activity (7.0 mol of deoxynucleotide incorporated in 30 min/mg of protein) and primase activities (13 mol of deoxynucleotide incorporated in 30 min/mg of protein, as performed by Schneider et al) (10), comparable with those of the pol ␣-primase complex isolated from extracts of S. pombe cells (data not shown). His-tagged spMcm10p (called Cdc23p in S. pombe) was cloned and expressed in E. coli and purified as described under "Materials and Methods."…”

Section: Resultssupporting

confidence: 53%

Primer Utilization by DNA Polymerase α-Primase Is Influenced by Its Interaction with Mcm10p

Fien

Cho

Lee

et al. 2004

Journal of Biological Chemistry

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Models of DNA replication in yeast andMuch progress has been made identifying proteins that assemble during early stages in replication prior to the start of DNA synthesis. These events are directed by origins of replication, which orchestrate formation of the pre-replication complex (pre-RC) 1 at origins during the G 1 phase of the cell cycle. They include origin binding by the origin recognition complex and subsequent recruitment of Cdc6 and Cdt1, followed by the loading of the Mcm2-7 complex (1, 2). Further progression is regulated by the cell cycle through the activation of two S phase promoting kinases, the S phase cyclin-dependent kinase and Cdc7/Dbf4 kinase. Activation of the pre-RC by these kinases promotes Cdc45 association with origins (3, 4), events that lead subsequently to DNA unwinding and the ordered assembly of the replication fork machinery.The DNA polymerase ␣-primase (pol ␣-primase) complex also plays an essential role in initiation by synthesizing short RNA primers required to begin both leading and lagging strand DNA synthesis. The four subunit structure of pol ␣-primase is conserved, and each subunit is essential for cell viability in yeast (5, 6). The largest subunit of pol ␣-primase (p180) contains the DNA polymerase catalytic center (7) and elongates RNA primers of 8 -12 nucleotides synthesized by primase. The catalytic center of primase is located within the p48 subunit (8), which exists as a tight complex with p58 (9, 10). The p58 subunit was shown to be required for distinct aspects of primer synthesis, including initiation and elongation (11). The fourth subunit of DNA pol ␣-primase, p70/␤, is suggested to recruit the complex onto chromatin as a result of its cell cycle-regulated phosphorylated state (12, 13). Polymerase ␣-primase subunits have been reported to interact with a number of replication proteins including Cdc45 (14), Dna2 (15), RPA (16,17), and viral initiator proteins (16,18,19), consistent with the idea that replication forks are large precisely assembled multiprotein complexes.Mcm10p has been implicated both in the initiation and elongation steps of DNA replication. MCM10 was first identified in Saccharomyces cerevisiae using screens for mutants defective in DNA replication and for the stable maintenance of plasmids (20,21). Initiation at replication origins is drastically reduced in the mcm10-1 mutant, and replication across origins is impeded (21,22). Mutations in MCM10 result in a delay in the completion of DNA synthesis after cells are released from HU arrest (23), suggesting that Mcm10p is essential for continued fork progression. MCM10 mutants are suppressed by mutant MCM5 and MCM7 genes and are synthetically lethal with mutant genes of ORC, CDC45, DNA2, DPB11, and genes encoding subunits of DNA polymerase ␦ and ⑀ (22-27). Seven new mutants named slm1-slm6 for synthetically lethal with mcm10 include mutations in genes that are allelic to MCM7, MCM2, CDC45, DNA2, and mutations in novel DNA repair genes represented by SLM2 and SLM6 (28). Biochemical and genetic int...

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Section: Resultssupporting

confidence: 53%

Primer Utilization by DNA Polymerase α-Primase Is Influenced by Its Interaction with Mcm10p

Fien

Cho

Lee

et al. 2004

Journal of Biological Chemistry

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“…Primase Activities of pol-prim-Primase activities of baculovirus-expressed pol-prim tetramer (p180-p68-p58-p48), and of bacterially expressed prim 2 (p58-p48) as well as p48 on poly(dC) and poly(dT) have been presented previously (13,21). To compare the initiation activities of the purified proteins ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, RPA, prim 2 , and the individual primase subunits p58 and p48 were bacterially expressed and purified as outlined before (13,57). Five pmol of each protein were adsorbed to Strataclean resin, and the beads were applied to SDS-polyacrylamide gel electrophoresis (58).…”

Section: Methodsmentioning

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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“…The recombinant GANP-PD was purified from bacteria harboring pET-ganp-PD after isopropyl-␤-D(Ϫ)-thiogalactopyranoside (IPTG) induction using His-Bind Resin (Novagen). Subsequently, the buffer used to dissolve the recombinant protein was replaced with 50 mM potassium phosphate (pH 7.5), 3 mM 2-mercaptoethanol, 0.1 mM MnSO 4 , and 1 mM MgCl 2 (15). For long storage, 10% glycerol was added.…”

Section: Generation Of Recombinant Primase Domain Of Ganp (Ganp-pd)mentioning

confidence: 99%

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Kuwahara

Tomiyasu

Fujimura

et al. 2001

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

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Primase Activity of Human DNA Polymerase α-Primase

Cited by 48 publications

References 47 publications

Primer Utilization by DNA Polymerase α-Primase Is Influenced by Its Interaction with Mcm10p

Primer Utilization by DNA Polymerase α-Primase Is Influenced by Its Interaction with Mcm10p

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

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

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