Pea chloroplast DNA primase: characterization and role in initiation of replication
A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase.
In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
Key wordschloroplasts DNA primase DNA replication Pisum sativum
Unable to display preview. Download preview PDF.
- 4.Chu NM, Shapiro DR, Oishi KK, Tewari KK: Distribution of transfer RNA genes in the Pisum sativum chloroplast DNA. Plant Mol Biol 4: 65–79 (1985).Google Scholar
- 6.Crouse EJ, Schmitt JM, Bohnert H-J: Chloroplast and cyanobacterial genomes, genes and RNAs: a compilation. Plant Mol Biol Rep 3: 43–89 (1985).Google Scholar
- 7.de Haas JM, Kool AJ, Overbeeke N, van Brug W, Nijkamp HJJ: Characterization of DNA synthesis and chloroplast DNA replication initiation in a Petunia hybrida chloroplast lysate system. Curr Genet 12: 377–386 (1987).Google Scholar
- 10.Hiratsuka J, Shimada H, Whittier R, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun C-R, Meng B-Y, Li Y-Q, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M: The complete sequence of the rice (Oryza sativa) chloroplast genome: Intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217: 185–194 (1989).PubMedGoogle Scholar
- 11.Jolly SO, Bogorad L: Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase. Proc Natl Acad Sci USA 77: 815–828 (1980).Google Scholar
- 15.Kornberg A: DNA Replication, pp. 384–396. W.H. Freeman, San Francisco (1980).Google Scholar
- 17.Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual, p. 478. Cold Spring Harbor Laboratory, cold Spring Harbor, NY (1982).Google Scholar
- 19.McKown RL, Tewari KK: Purification and properties of a pea chloroplast DNA polymerase. Proc Natl Acad Sci USA 81: 2354–2358 (1984).Google Scholar
- 22.Nielsen BL, Tewari KK: Pea chloroplast topoisomerase I: purification, characterization, and role in replication. Plant Mol Biol 11: 3–14 (1988).Google Scholar
- 23.Nielsen BL, Meeker R, Tewari KK: Replication of chloroplast DNA: Replication origins, topoisomerase I, and in vitro replication. In: Singhal GS, Barber J, Dilley RA, Govindjee, Haselkorn R, Mohanty P (eds) Photosynthesis: Molecular Biology and Bioenergetics, pp. 37–52. Narosa Publishing House, New Delhi, India/Springer Verlag, Berlin (1989).Google Scholar
- 25.Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H: Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574 (1986).Google Scholar
- 30.Ravel-Chapuis P, Heizmann P, Nigon V: Electron microscopic localization of the replication origin of Euglena gracilis chloroplast DNA. Nature 300: 78–81 (1982).Google Scholar
- 34.Shapiro DR, Tewari KK: Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA. Plant Mol Biol 6: 1–12 (1986).Google Scholar
- 35.Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Suguira M: The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043–2049 (1986).Google Scholar
- 36.Sun E, Shapiro DR, Wu B-W, Tewari KK: Specific in vitro transcription of 16S rRNA gene by pea chloroplast RNA polymerase. Plant Mol Biol 6: 429–439 (1986).Google Scholar
- 38.Tewari KK, Goel A: Solubilization and partial purification of RNA polymerase from pea chloroplasts. Biochemistry 22: 2142–2148 (1983).Google Scholar
- 50.Wu M, Lou JK, Chang DY, Chang CH, Nie ZQ: Structure and function of a chloroplast DNA replication origin of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 83: 6761–6765 (1986).Google Scholar