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. 1998 Nov;118(3):1029-38.
doi: 10.1104/pp.118.3.1029.

A Chloroplast DNA Helicase II From Pea That Prefers Fork-Like Replication Structures

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Free PMC article

A Chloroplast DNA Helicase II From Pea That Prefers Fork-Like Replication Structures

N Tuteja et al. Plant Physiol. .
Free PMC article

Abstract

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5'-tailed fork was more active than the 3'-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3' to 5' along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mM KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 &mgr;M and 2.2 &mgr;M, respectively) and ATPase (Ki approximately 1.3 &mgr;M and 3.0 &mgr;M, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

Figures

Figure 1
Figure 1
Purification scheme of pea ctDNA helicase II. The strategy used for fractionation of DNA helicase II is shown. The numbers indicate the molarity of the KCl gradient or step elution to elute the enzyme activity.
Figure 2
Figure 2
SDS-PAGE of purified pea ctDNA helicase II. Fraction VI (60 ng, lane 1) and the Mr marker (lane 2) were separated on a 12% polyacrylamide gel and visualized by silver staining.
Figure 3
Figure 3
Effect of ATP (A), MgCl2 (B), and KCl (C) on pea ctDNA helicase II activity. In each reaction 5 ng of fraction VI with 1 ng of the 5′-tailed substrate was used with varying concentrations of ATP, MgCl2, or KCl. Quantitative data are displayed on the right side of each autoradiogram. The structure of the substrate is shown on the left side of each gel. Asterisks denote the 32P-labeled end. Lanes marked “No enzyme” and “Heated” are the reactions without the enzyme and with heat-denatured substrates, respectively. The activity is shown as percent unwinding.
Figure 4
Figure 4
Preference of nucleotides for pea ctDNA helicase II activity. The standard helicase reactions were performed with 5 ng of fraction VI, 1 ng of 5′-tailed substrate, and 2 mm NTP or deoxyribonucleoside triphosphate. The amount of unwound DNA was quantitated and plotted as a histogram above the autoradiogram of the gel. Lanes 1 and 10 are the reactions without the enzyme and with the heat-denatured substrate, respectively. Lanes 2 to 9 are reactions in the presence of ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, and dTTP, respectively. The structure of the substrate is shown on the left side of the autoradiogram. The asterisk denotes the 32P-labeled end.
Figure 5
Figure 5
Kinetics and concentration dependence of pea ctDNA helicase II. The enzyme activity data from the autoradiograms (left) were quantitated and are shown on the right. The structure of the substrate used is shown on the extreme left. Asterisks denote the 32P-labeled end. A, The standard reaction was carried out with 5 ng of fraction VI at the times indicated. B, An increasing amount of fraction VI was used in the standard helicase assay. The concentrations are indicated at the top of each lane. Lanes labeled “No enzyme” and “Heated” are the reactions without the enzyme and with the heat-denatured substrate, respectively.
Figure 6
Figure 6
Preference of forked DNA structures for unwinding activity of pea ctDNA helicase II. The DNA helicase reactions were performed under standard conditions using different DNA substrates that contained either no tail (A), a 3′ tail (B), a 5′ tail (C), or both 3′ and 5′ tails (D and E). Small linear substrates with forked structures, which also represent 3′ to 5′ (F) or 5′ to 3′ (G) direction specificity, and a blunt-ended DNA substrate (H) were also used. The schematic structure of each substrate is shown on the left side of the autoradiogram of the gel. Asterisks denote the 32P-labeled end. The percent unwinding is shown at the top of each panel. In each panel, lane 1 is the reaction without the enzyme, lane 2 is the reaction with the enzyme (5 ng), and lane 3 is the heat-denatured substrate.
Figure 7
Figure 7
Direction of unwinding by pea ctDNA helicase II. The structure of the linear substrate for the 3′ to 5′ direction (A) and 5′ to 3′ direction (B) is shown on top of the autoradiogram. In each gel, lane 1 is the reaction without enzyme; lane 2 is the reaction with 8.5 ng of fraction VI; and lane 3 is the heat-denatured substrate. Asterisks denote 32P-labeled ends.
Figure 8
Figure 8
Effect of DNA-interacting ligands on DNA unwinding (A) and ATPase (B) activities of pea ctDNA helicase II. The standard helicase reaction was performed with 5 ng of fraction VI, 1 ng of the 5′-tailed substrate, and 50 μm of the compound. The name of each compound is located on top of each autoradiogram.
Figure 9
Figure 9
Titration of inhibition of unwinding activity of pea ctDNA helicase II by daunorubicin (A) and nogalamycin (B). The DNA helicase reactions were performed in the presence of increasing concentrations of the ligand using 1 ng of 32P-labeled substrate and 5 ng of the pure enzyme. The quantitative curve is shown on the left side of each autoradiogram. Various concentrations of each ligand used are located at the top of each lane.
Figure 10
Figure 10
Titration of inhibition of DNA-dependent ATPase activity of pea ctDNA helicase II by daunorubicin (A) and nogalamycin (B). The standard ATPase reactions were performed in the presence of increasing concentrations of the ligand using 5 ng of the pure enzyme. The quantitative curve is shown on left side of each autoradiogram of the TLC plate. The positions of the Pi and ATP spots are indicated by arrows. Various concentrations of each ligand used are indicated at the top of each lane.

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