The 68 kDa calmodulin-binding protein is tightly associated with the multiprotein DNA polymerase .alpha.-primase complex in HeLa cells

Biochemistry ◽  
1995 ◽  
Vol 34 (12) ◽  
pp. 3878-3883 ◽  
Author(s):  
Qui Ping Cao ◽  
Claire A. McGrath ◽  
Earl F. Baril ◽  
Peter J. Quesenberry ◽  
G. Prem Veer Reddy
Keyword(s):  
Dna Polymerase ◽  
Hela Cells ◽  
Binding Protein ◽  
Dna Polymerase Alpha ◽  
Calmodulin Binding ◽  
Primase Complex

Binding of the DNA polymerase .alpha.-DNA primase complex to the nuclear matrix in HeLa cells

Biochemistry ◽  
1987 ◽  
Vol 26 (18) ◽  
pp. 5600-5607 ◽  
Author(s):  
James M. Collins ◽  
Annie K. Chu
Keyword(s):  
Dna Polymerase ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Dna Polymerase Alpha ◽  
Dna Primase ◽  
Primase Complex

In vitro replication of DNA containing either the SV40 or the polyoma origin

1987 ◽  
Vol 317 (1187) ◽  
pp. 439-453 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Hela Cells ◽  
Dna Binding Protein ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Origin ◽  
Sv40 Dna ◽  
Primase Complex

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro . Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo . The host-cell source of DNA polymerase α - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase α - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).

scholarly journals Subunits of the DNA polymerase alpha‐primase complex promote Notch‐mediated proliferation with discrete and shared functions in C. elegans germline

FEBS Journal ◽  
2018 ◽  
Vol 285 (14) ◽  
pp. 2590-2604 ◽  
Author(s):  
Dong Suk Yoon ◽  
Dong Seok Cha ◽  
Mohammad A. Alfhili ◽  
Brett D. Keiper ◽  
Myon‐Hee Lee
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Alpha ◽  
C Elegans ◽  
Primase Complex

scholarly journals Eukaryotic DNA primase appears to act as oligomer in DNA-polymerase-alpha-primase complex

1992 ◽  
Vol 206 (1) ◽  
pp. 7-13 ◽  
Author(s):  
Vladimir N. PODUST ◽  
Olga V. VLADIMIROVA ◽  
Elena N. MANAKOVA ◽  
Olga I. LAVRIK
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerase Alpha ◽  
Dna Primase ◽  
Eukaryotic Dna ◽  
Primase Complex

scholarly journals DNA polymerase α-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini

1994 ◽  
Vol 298 (3) ◽  
pp. 529-535
Author(s):  
S Niranjanakumari ◽  
K P Gopinathan
Keyword(s):  
Dna Polymerase ◽  
Larval Instar ◽  
Polymerase Activity ◽  
Dna Polymerase Delta ◽  
Philosamia Ricini ◽  
Dna Polymerase Alpha ◽  
Silk Glands ◽  
Tight Association ◽  
Mulberry Silkworm ◽  
Primase Complex

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system

1990 ◽  
Vol 232 (2) ◽  
pp. 141-153 ◽  
Author(s):  
Michael P. Carty ◽  
Yukio Ishimi ◽  
Arthur S. Levine ◽  
Kathleen Dixon
Keyword(s):  
Dna Polymerase ◽  
Hela Cells ◽  
High Fidelity ◽  
Replication System ◽  
Dna Polymerase Alpha

scholarly journals The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication.

1994 ◽  
Vol 14 (2) ◽  
pp. 923-933 ◽  
Author(s):  
M Foiani ◽  
F Marini ◽  
D Gamba ◽  
G Lucchini ◽  
P Plevani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Chromosomal Dna ◽  
Dna Polymerase Alpha ◽  
B Subunit ◽  
Primase Complex

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.

scholarly journals Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex.

Genetics ◽  
1993 ◽  
Vol 133 (2) ◽  
pp. 183-191 ◽  
Author(s):  
M P Longhese ◽  
L Jovine ◽  
P Plevani ◽  
G Lucchini
Keyword(s):  
Dna Polymerase ◽  
Large Subunit ◽  
Spontaneous Mutation ◽  
Critical Role ◽  
Direct Interaction ◽  
Small Subunit ◽  
Dna Polymerase Alpha ◽  
Dna Primase ◽  
Primase Complex

Abstract Different pri1 and pri2 conditional mutants of Saccharomyces cerevisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and error-prone repair, due to defective DNA replication in the primase mutants. Furthermore, pri1 and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri1 and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase alpha, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase alpha-primase complex.

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