Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha.

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.

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DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.79.23.7209 ◽

1982 ◽

Vol 79 (23) ◽

pp. 7209-7213 ◽

Cited By ~ 50

Author(s):

M. Shioda ◽

E. M. Nelson ◽

M. L. Bayne ◽

R. M. Benbow

Keyword(s):

Xenopus Laevis ◽

Dna Polymerase ◽

Dna Polymerase Alpha ◽

Dna Primase

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

Genetics ◽

10.1093/genetics/133.2.183 ◽

1993 ◽

Vol 133 (2) ◽

pp. 183-191 ◽

Cited By ~ 3

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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