Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle.

Miyazawa, Hiroshi; Izumi, Masako; Tada, Shusuke; Takada, R; Masutani, Mitsuko; Ui, Michio; Hanaoka, Fumio

doi:10.1016/s0021-9258(18)53069-6

Cited by 73 publications

(10 citation statements)

References 70 publications

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“…DNA pol R /DNA primase form a complex of four subunits, the largest of which is the catalytic subunit (∼180 kDa) (Wong et al, 1986), and the two smallest subunits comprise the DNA primase (∼58-and ∼48 kDa) (Bambara and Jessee, 1991). When quiescent cells are stimulated to proliferate, mRNA levels of all four subunits increase simultaneously prior to DNA synthesis; thus, transcription of the genes for DNA pol R and DNA primase are likely regulated by a common mechanism (Miyazawa et al, 1993). Expression of DNA pol R, DNA primase, and RPA were significantly reduced in PARP-depleted antisense cells (Figure 7), suggesting that PARP may be implicated in the expression of the corresponding genes during entry of cells into S phase.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the Expression or Recruitment of Components of the DNA Synthesome by Poly(ADP-Ribose) Polymerase

et al. 1998

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Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.

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Section: Discussionmentioning

confidence: 99%

Regulation of the Expression or Recruitment of Components of the DNA Synthesome by Poly(ADP-Ribose) Polymerase

et al. 1998

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“…For synchronization, cells were cultured in DMEM supplemented with 0.4% fetal calf serum and non-essential amino acids for 72 h, and then released by changing the medium to DMEM containing 15% fetal calf serum and non-essential amino acids. Poly(A) + RNA was isolated every 4 h and used for northern blotting as described (29). To monitor DNA synthesis, cells were pulse-labeled with BrdU for 20 min, and incorporated BrdU was quantified by enzyme-linked immunosorbent assay using the BrdU labeling detection kit III (Boehringer Mannheim).…”

Section: Northern Blot Analysismentioning

confidence: 99%

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G2 phase

Izumi¹

2000

Nucleic Acids Research

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Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

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“…Two-codon insertion mutagenesis and deletion analysis of the POL12 gene. The amino acid sequence derived from the largest POL12 ORF shows significant homology with those of the corresponding subunits of the Pol a-primase complex from human, mouse, and Drosophila melanogaster cells (14,15,43). Clusters of conserved amino acid residues are mostly confined within the COOH-terminal two-thirds of these proteins (14).…”

Section: Resultsmentioning

confidence: 99%

The B Subunit of the DNA Polymerase α-Primase Complex in Saccharomyces cerevisiae Executes an Essential Function at the Initial Stage of DNA Replication

Foiani¹,

Marini²,

Gamba

et al. 1994

Molecular and Cellular Biology

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The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.

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Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle.

Cited by 73 publications

References 70 publications

Regulation of the Expression or Recruitment of Components of the DNA Synthesome by Poly(ADP-Ribose) Polymerase

Regulation of the Expression or Recruitment of Components of the DNA Synthesome by Poly(ADP-Ribose) Polymerase

The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G2 phase

The B Subunit of the DNA Polymerase α-Primase Complex in Saccharomyces cerevisiae Executes an Essential Function at the Initial Stage of DNA Replication

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