Purification and properties of a DNA primase from Nicotianatabacum

Abstract

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase alpha-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase.

Maize replicative α‐type DNA polymerase: separation of polymerase and primase activities and recognition of primase subunits

“…1994). This 68 kDa size is somewhat larger than the 60 kDa wheat primase and the 50 kDa Nicotiana primase (Laquel et al. 1994, García‐Maya and Buck 1998).…”

“…The 8000‐fold purified pol 2 contains a tightly bound DNA primase activity that incorporates ribonucleotides into ssDNA templates (García et al. 1997), which share some characteristics with yeast, animal, wheat and Nicotiana primases (Wang et al. 1984, Jaswinsky and Edellman 1985, Laquel et al.…”

“…This 90 kDa size for DNA pol 2 could represent a proteolytic fragment of the complete enzyme, according to reported sizes for a-type polymerase catalytic subunits, including a plant enzyme (Kornberg andBaker 1992, Yokoi et al 1997). The 8000-fold purified pol 2 contains a tightly bound DNA primase activity that incorporates ribonucleotides into ssDNA templates (García et al 1997), which share some characteristics with yeast, animal, wheat and Nicotiana primases (Wang et al 1984, Jaswinsky and Edellman 1985, Laquel et al 1990, García-Maya and Buck 1998. DNA primases reported to date contain two polypeptides of around 58 and 49 kDa (Lehman and Kaguni 1989).…”

Multiple Functions of DNA Polymerases

“…Early biochemical work resulted in the isolation of several polypeptides with polymerase activity and properties that were Downloaded by [New York University] at 05:55 06 October 2014 similar to that of their animal counterparts Tarrago-Litvak et al, 1975;Coello and Vazquez-Ramos, 1995;Seto et al, 1998). Among these, several polypeptides with primase activity were identified in a wide variety of organisms (Litvak et al, 1984;Nielsen et al, 1991;Laquel et al, 1994;Garcia-Maya and Buck, 1998). The analysis of different sequenced plant genomes suggests that, like in other eukaryotes, this activity corresponds to a Polα/primase protein complex, and that initiation of replication shares the same components in plants as in other eukaryotes.…”

Application of capillary electrophoresis for DNA-protein binding tests

“…In this context studies directed towards identification,, isolation and characterization of DSB or SSB from different tissues or organisms are still a current research task (Kaiserman etaL 1989, Kenny etal. 1990, Kenny et al 1994, van der Knaap et al 1997, Garcia-Maya and Buck 1998 A routine procedure for identification of proteins with specificity for DNA combines affinity chromatography and electrophoretic gel mobility-shift assay (EMSA, Mitas et al 1997, Revers et al 1999. The DNA-protein complexes are detectable as more slowly migrating fractions, in comparison with free probes.…”