scholarly journals Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori

1991 ◽  
Vol 201 (2) ◽  
pp. 431-438 ◽  
Author(s):  
Somashekarappa NIRANJANAKUMARI ◽  
Karumathil P. GOPINATHAN
Keyword(s):  
Bombyx Mori ◽  
Dna Polymerase ◽  
Dna Polymerase Alpha ◽  
Silk Glands ◽  
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.

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 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.

scholarly journals Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase.

1991 ◽  
Vol 11 (4) ◽  
pp. 2108-2115 ◽  
Author(s):  
K L Collins ◽  
T J Kelly
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Dna Polymerase Alpha ◽  
Primase Complex

Studies of simian virus 40 (SV40) DNA replication in a reconstituted cell-free system have established that T antigen and two cellular replication proteins, replication protein A (RP-A) and DNA polymerase alpha-primase complex, are necessary and sufficient for initiation of DNA synthesis on duplex templates containing the SV40 origin of DNA replication. To better understand the mechanism of initiation of DNA synthesis, we analyzed the functional interactions of T antigen, RP-A, and DNA polymerase alpha-primase on model single-stranded DNA templates. Purified DNA polymerase alpha-primase was capable of initiating DNA synthesis de novo on unprimed single-stranded DNA templates. This reaction involved the synthesis of a short oligoribonucleotide primer which was then extended into a DNA chain. We observed that the synthesis of ribonucleotide primers by DNA polymerase alpha-primase is dramatically stimulated by SV40 T antigen. The presence of T antigen also increased the average length of the DNA product synthesized on primed and unprimed single-stranded DNA templates. These stimulatory effects of T antigen required direct contact with DNA polymerase alpha-primase complex and were most marked at low template and polymerase concentrations. We also observed that the single-stranded DNA binding protein, RP-A, strongly inhibits the primase activity of DNA polymerase alpha-primase, probably by blocking access of the enzyme to the template. T antigen partially reversed the inhibition caused by RP-A. Our data support a model in which DNA priming is mediated by a complex between T antigen and DNA polymerase alpha-primase with the template, while RP-A acts to suppress nonspecific priming events.

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.

Replication factor A is required in vivo for DNA replication, repair, and recombination

1994 ◽  
Vol 14 (12) ◽  
pp. 7884-7890
Author(s):  
M P Longhese ◽  
P Plevani ◽  
G Lucchini
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Binding Protein ◽  
Dna Polymerase Delta ◽  
Gene Encoding ◽  
Replication Factor ◽  
Dna Polymerase Alpha ◽  
Essential Step ◽  
Primase Complex

Replication factor A (RF-A) is a heterotrimeric single-stranded-DNA-binding protein which is conserved in all eukaryotes. Since the availability of conditional mutants is an essential step to define functions and interactions of RF-A in vivo, we have produced and characterized mutations in the RFA1 gene, encoding the p70 subunit of the complex in Saccharomyces cerevisiae. This analysis provides the first in vivo evidence that RF-A function is critical not only for DNA replication but also for efficient DNA repair and recombination. Moreover, genetic evidence indicate that p70 interacts both with the DNA polymerase alpha-primase complex and with DNA polymerase delta.

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