Pea chloroplast DNA primase: characterization and role in initiation of replication

Abstract

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.

Biochemical properties of a plastidial DNA polymerase of rice

“…A previous study showed that a primase activity is present in chloroplasts as well as nuclei (Nielsen et al 1991). In addition, according to the rice genome database, rice cells contain the homolog of phage T7 gene 4 protein, which has primase and helicase domains (GenBank accession number AP005610).…”

The transcriptional apparatus of chloroplasts

“…The latter has been further purified and characterized as a 115 to 120 kDa protein that can synthesize RNA primers of the size range from 4 to 60 nucleotides when single-stranded DNA is used as a template. The DNA primase is insensitive against heparin, tagetitoxin, and antibodies specific for RNA polymerase subunits, 54 which also shows that this activity is not due to a catalytic subunit that may have separated from RNA polymerase.…”

The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?

“…A dependence of template activity on supercoiling has been reported for partially purified cp RNA polymerase activities (18,19). The determination of the size of the transcripts is important to rule out the possibility that the 110-kDa polypeptide represents a DNA primase, because it has been shown that DNA primase of pea chloroplasts sediments as a 115-to 120-kDa polypeptide on glycerol gradients (20). Transcription activity with this template was measured by gel electrophoresis (Fig.…”