Replication of Chloroplast DNA: Replication Origins, Topoisomerase I, and In Vitro Replication
Replication origins in pea chloroplast DNA (ctDNA) have been mapped in the 44 kbp SalI fragment A. Restriction digests of pea ctDNA with SmaI, PstI and SalI/SmaI have allowed orientation of the two D-loops on the restriction map. One D-loop is located in the spacer region between the 16S and 23S rRNA genes, and the second D-loop is located downstream of the 23S rRNA gene, about 6 kbp apart from the first D-loop. Denaturation mapping of two recombinants, pCP12–7 and pCBI-12, which contain the two D-loops, confirmed the location of the D-loops. Denaturation mapping studies also showed that the D-loop present in the rRNA spacer region has a much higher A+T content than the other D-loop.
Using a partially purified pea chloroplast replication system, the supercoiled pCP12–7 and pCB1–12 recombinants served as highly active templates for DNA synthesis. Some smaller subclones of the A+T-rich D-loop region also show activity, including a 1.2 kbp BamHI-HindIII clone. A smaller 700 bp BamHI-BglII clone shows very low activity suggesting that sequences outside the BglII region are important for replication. Analysis of the DNA synthesised in vitro from these templates showed that full-length DNA was made, while other recombinants from other regions of the pea ctDNA showed no significant activity in vitro.
Pea chloroplast topoisomerase I was purified 5000-fold to homogeneity, and was found to consist of a single polypeptide of 112,000 daltons. The enzyme catalyses an ATP-independent relaxation of negatively supercoiled DNA. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. These properties support the identification of the pea chloroplast enzyme as a prokaryotic-like topoisomerase I.
The role of topoisomerase I in the replication of ctDNA has been established by using a partially purified pea chloroplast replication system which lacks topoisomerase I activity, and recombinant templates which contain the pea ctDNA replication origins. A 2–6-fold stimulation of DNA synthesis resulted when the purified topoisomerase I was added to reactions at 80 mM KCl. When the reactions were carried out at 125 mM KCl, which is completely inhibitory for topoisomerase I but does not affect non-specific DNA polymerase activity, topoisomerase I addition caused no stimulation of activity. Novobiocin, an inhibitor of topoisomerase II, was found to have no effect on the in vitro replication of the ctDNA recombinants.
KeywordsReplication Origin Replication System Cumulative Histogram Heparin Sepharose Column Denaturation Loop
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