Abstract
The recently discovered eukaryotic primases have been found in tight association with certain DNA polymerase alpha forms. Here I present evidence that the high mol. wt. catalytic polypeptide (125,000) of an apparently homogeneous DNA polymerase alpha from freshly harvested calf thymus contains both polymerase and primase activity. This conclusion derives from the following three facts: (1) the two enzyme activities cannot be separated upon velocity sedimentation in 1.7 M urea, (2) both activities elute at a pI of 5.25 upon chromatofocussing and (3) after SDS-electrophoresis, renaturation of the enzymes in situ and measurement of DNA polymerase and primase activities in the gels, both enzymes have identical mobilities and coincide with the high mol. wt. catalytic subunit of DNA polymerase alpha.
Full text
PDF![133](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d1b2/555100/42127ff7dcc4/emboj00254-0127.png)
![134](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d1b2/555100/f2cf654635d9/emboj00254-0128.png)
![135](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d1b2/555100/a02e8b945d33/emboj00254-0129.png)
![136](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d1b2/555100/120cddcc9cce/emboj00254-0130.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Albert W., Grummt F., Hübscher U., Wilson S. H. Structural homology among calf thymus alpha-polymerase polypeptides. Nucleic Acids Res. 1982 Feb 11;10(3):935–946. doi: 10.1093/nar/10.3.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Conaway R. C., Lehman I. R. A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2523–2527. doi: 10.1073/pnas.79.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eliasson R., Reichard P. Primase initiates Okazaki pieces during polyoma DNA synthesis. Nature. 1978 Mar 9;272(5649):184–185. doi: 10.1038/272184a0. [DOI] [PubMed] [Google Scholar]
- Grosse F., Krauss G. Purification and partial characterization of a DNA polymerase alpha species from calf thymus. Nucleic Acids Res. 1980 Dec 11;8(23):5703–5714. doi: 10.1093/nar/8.23.5703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grosse F., Krauss G. Purification of a 9S DNA polymerase alpha species from calf thymus. Biochemistry. 1981 Sep 15;20(19):5470–5475. doi: 10.1021/bi00522a019. [DOI] [PubMed] [Google Scholar]
- Grummt F., Waltl G., Jantzen H. M., Hamprecht K., Huebscher U., Kuenzle C. C. Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6081–6085. doi: 10.1073/pnas.76.12.6081. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holmes A. M., Hesslewood I. P., Johnston I. R. Evidence that DNA polymerase-alpha of calf thymus contains a subunit of molecular weight 155000. Eur J Biochem. 1976 Feb 16;62(2):229–235. doi: 10.1111/j.1432-1033.1976.tb10152.x. [DOI] [PubMed] [Google Scholar]
- Hübscher U., Gerschwiler P., McMaster G. K. A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates. EMBO J. 1982;1(12):1513–1519. doi: 10.1002/j.1460-2075.1982.tb01348.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hübscher U., Kornberg A. The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6284–6288. doi: 10.1073/pnas.76.12.6284. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hübscher U., Kuenzle C. C., Spadari S. Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation. Nucleic Acids Res. 1977 Aug;4(8):2917–2929. doi: 10.1093/nar/4.8.2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hübscher U., Spanos A., Albert W., Grummt F., Banks G. R. Evidence that a high molecular weight replicative DNA polymerase is conserved during evolution. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6771–6775. doi: 10.1073/pnas.78.11.6771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaufmann G., Falk H. H. An oligoribonucleotide polymerase from SV40-infected cells with properties of a primase. Nucleic Acids Res. 1982 Apr 10;10(7):2309–2321. doi: 10.1093/nar/10.7.2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozu T., Yagura T., Seno T. De novo DNA synthesis by a novel mouse DNA polymerase associated with primase activity. Nature. 1982 Jul 8;298(5870):180–182. doi: 10.1038/298180a0. [DOI] [PubMed] [Google Scholar]
- Lichy J. H., Field J., Horwitz M. S., Hurwitz J. Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5225–5229. doi: 10.1073/pnas.79.17.5225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Masaki S., Koiwai O., Yoshida S. 10 S DNA polymerase alpha of calf thymus shows a microheterogeneity in its large polypeptide component. J Biol Chem. 1982 Jun 25;257(12):7172–7177. [PubMed] [Google Scholar]
- Riedel H. D., König H., Stahl H., Knippers R. Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity. Nucleic Acids Res. 1982 Sep 25;10(18):5621–5635. doi: 10.1093/nar/10.18.5621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rowen L., Kornberg A. A ribo-deoxyribonucleotide primer synthesized by primase. J Biol Chem. 1978 Feb 10;253(3):770–774. [PubMed] [Google Scholar]
- Sadowski P. D., Hurwitz J. Enzymatic breakage of deoxyribonucleic acid. II. Purification and properties of endonuclease IV from T4 phage-infected Escherichia coli. J Biol Chem. 1969 Nov 25;244(22):6192–6198. [PubMed] [Google Scholar]
- Scherzinger E., Lanka E., Morelli G., Seiffert D., Yuki A. Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication. Eur J Biochem. 1977 Feb;72(3):543–558. doi: 10.1111/j.1432-1033.1977.tb11278.x. [DOI] [PubMed] [Google Scholar]
- Spanos A., Sedgwick S. G., Yarranton G. T., Hübscher U., Banks G. R. Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Apr 24;9(8):1825–1839. doi: 10.1093/nar/9.8.1825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tseng B. Y., Ahlem C. N. DNA primase activity from human lymphocytes. Synthesis of oligoribonucleotides that prime DNA synthesis. J Biol Chem. 1982 Jul 10;257(13):7280–7283. [PubMed] [Google Scholar]
- Villani G., Sauer B., Lehman I. R. DNA polymerase alpha from Drosophila melanogaster embryos. Subunit structure. J Biol Chem. 1980 Oct 10;255(19):9479–9483. [PubMed] [Google Scholar]
- Yagura T., Kozu T., Seno T. Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization. J Biochem. 1982 Feb;91(2):607–618. doi: 10.1093/oxfordjournals.jbchem.a133732. [DOI] [PubMed] [Google Scholar]
- Yagura T., Kozu T., Seno T. Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size. J Biol Chem. 1982 Sep 25;257(18):11121–11127. [PubMed] [Google Scholar]