Figure 1.
Comparison of amino acid sequence of Plasmodium falciparum (Pf) UvrD (1–1441) and Escherichia coli (E. coli) UvrD (1–720).
The alignment was done using BLAST program (http://blast.ncbi.nlm.nih.gov/Blast). The conserved motifs are boxed in red color and the name of each motif (from Q to VIa) is written in roman numerals. The PlasmoDB number for P. falciparum UvrD sequence is PFE0705c and the accession number for E. coli UvrD sequence is BAA00048.1. The black, blue and purple dotted lines indicate the PfUDN, PfUDC1 and PfUDC2 fragments respectively. The orange box indicates the domain 1A (from amino acid 1–722) and the blue box inside it indicates the domain 1B (from amino acid 150–464). The purple box denotes the domain 2A (from amino acid 723–1441) and the green box inside it indicates the domain 2B (from amino acid 896–1359).
Figure 2.
Schematic diagrams showing the domain organization. A,
E. coli and B, P. falciparum UvrD helicases. Domain analysis was done using Scan Prosite at (http://expasy.org). The domain structure was taken from the results and used in the figures. UvrD helicase ATP binding and UvrD C-terminal domains are shown. The numbers show the amino acids spanning these motifs. C, The detailed domain organization of P. falciparum UvrD helicase. The conserved sequences of each domain are written inside the boxes. The text in blue refers to the names of various conserved domains and the numbers refer to the amino acids separating the various domains and the length of N- and C-terminal extensions. This figure is not drawn to scale. D–F, The detailed domain structure of PfUDN, PfUDC1 and PfUDC2 fragments of P. falciparum UvrD. The details are as in C. The colored lines are same as in Figure 1 and correspond to domain 1A, 1B, 2A and 2B present in PfUDN, PfUDC1 and PfUDC2 respectively. (G–I) Structure modeling. The PfUvrD full-length sequence was submitted to Swissmodel server and the structure was obtained. The molecular graphic images were produced using the UCSF Chimera package from the resource for Biocomputing, Visualization, and Informatics (http://www.cgl.ucsf.edu/chimera) at the University of California, San Francisco (supported by NIH P41 RR-01081). G. Template; H. full-length PfUvrD; I. superimposed image.
Figure 3.
Purification and ATPase activity analysis.
A, Silver-stained gel of purified PfUDN. Lane M contains the protein molecular weight marker and lane 1 and 2 contain 0.3 and 0.2 µg of the purified PfUDN. B, Silver-stained gel of purified PfUDC1. Lane M contains the protein molecular weight marker and lane 1 contains 0.2 µg of the purified PfUDC1. C, Silver-stained gel of purified PfUDC2. Lane M contains the protein molecular weight marker and lane 1 contains 0.2 µg of the purified PfUDC2. D, Western blot of purified PfUDN. Lane M contains the protein molecular weight marker and lane 1 and 2 contain 0.3 and 0.2 µg of the purified PfUDN. E, Western blot of purified PfUDC1. Lane M contains the protein molecular weight marker and lane 1 contains 0.2 µg of the purified PfUDC1. F, Western blot of purified PfUDC2. Lane M contains the protein molecular weight marker and lane 1 contains 0.2 µg of the purified PfUDC2. G, ATPase activity of purified PfUDN. Lane C, reaction without enzyme, Lanes 1–4, reactions with enzyme in the presence of ssDNA and Mg2+. H, The quantitative data of the autoradiogram in G. I, Time dependence of ATPase activity of PfUDN. The time of incubation in minutes is mentioned at the top of the autoradiogram and C is the control reaction without enzyme. J, The quantitative data of the autoradiogram in I. K, ATPase activity of purified PfUDC1. Lane C, reaction without enzyme, Lanes 1–4, reactions with enzyme in the presence of ssDNA and Mg2+. L, The quantitative data of the autoradiogram in K. M, Time dependence of ATPase activity of PfUDC1. The time of incubation in minutes is mentioned at the top of the autoradiogram and C is the control reaction without enzyme. N, The quantitative data of the autoradiogram in M.
Figure 4.
A, Helicase activity of purified PfUDN, lanes 1–7 contain increasing concentration of PfUDN, C is reaction without enzyme and B is heat denatured substrate. B, The quantitative enzyme activity data from the autoradiogram in A are shown and the concentration of PfUDN is also written. C, Helicase activity of purified PfUDC1, lanes 1–4 contain increasing concentration of PfUDC1, C is reaction without enzyme and B is heat denatured substrate. D, The quantitative enzyme activity data from the autoradiogram in C are shown and the concentration of PfUDC1 is also written. E, Helicase activity of purified PfUDC2, lanes 1–3 contain increasing concentration of PfUDC2, C is reaction without enzyme and B is heat denatured substrate.
Figure 5.
Immunodepletion of ATPase and helicase activities.
A and B, Quantitative data of immunodepletion of ATPase activity. A, Lanes 1–3, reactions with increasing concentration of PfUDN pretreated with immune IgG and lanes 4–6, reactions with increasing concentration of PfUDN pretreated with pre-immune IgG. B, Lanes 1–3, reactions with increasing concentration of PfUDC1 pretreated with anti-his IgG and lanes 4–6, reactions with increasing concentration of PfUDC1 pretreated with pre-immune IgG. Each bar represents the mean percentage ± SD of two different experiments. C and D, Quantitative data of immunodepletion of helicase activity. C, Lanes 1–3, reactions with increasing concentration of PfUDN pretreated with immune IgG and lanes 4–6, reactions with increasing concentration of PfUDN pretreated with pre-immune IgG. D, Lanes 1–3, reactions with increasing concentration of PfUDC1 pretreated with anti-his IgG and lanes 4–6, reactions with increasing concentration of PfUDC1 pretreated with pre-immune IgG. Each bar represents the mean percentage ± SD of two different experiments.
Figure 6.
Kinetics of helicase activity.
Km and Vmax of helicase activity of A, PfUDN and B, PfUDC1. The helicase reactions were carried out as reported in materials and methods section and Km and Vmax were calculated from the plot.
Figure 7.
Further characterization of helicase activity.
A, Nucleotide requirement of helicase activity of PfUDN. Helicase activity of PfUDN in the presence of lane 1, dCTP, lane 2, CTP, lane 3, dGTP, lane 4, GTP, lane 5, dATP, lane 6, ATP, lane 7, dTTP and lane 8, UTP. Lane E is enzyme reaction of PfUDN in the absence of any NTP or dNTP. C1 and C2 are reactions without enzyme and B1 and B2 are heat denatured substrates respectively. B, The quantitative enzyme activity data from the autoradiogram in A are shown and the various NTPs/dNTPs used are also written. C, Helicase activity of PfUDN using varying concentration of ATP. Lane 1, 0.5, lane 2, 1.0, lane 3, 1.5, lane 4, 2.0, lane 5, 2.5 and lane 6, 5.0 mM ATP. C1 and C2 are reactions without enzyme and B is heat denatured substrate respectively. Lane E is enzyme reaction in the absence of ATP. D, The quantitative enzyme activity data from the autoradiogram in C are shown and the concentration of ATP used is also written. E, Unwinding activity of PfUDN with blunt end duplex substrate. The helicase reaction was performed under standard assay conditions; the structure of the substrate used and the autoradiogram of the gel are shown. Asterisk (*) denotes the 32P-labeled end. Lanes 1–6 are the reactions with increasing concentration of enzyme, C is no enzyme control and B is heat-denatured substrate, respectively. F, The quantitative enzyme activity data from the autoradiogram in E are shown and the concentration of PfUDN used is also written.
Figure 8.
Direction of unwinding by PfUDN.
A, The structures of the linear substrate for the 5′ to 3′ direction (substrate 3A, Tale S1). An asterisk (*) denotes the 32P-labeled end. B, Helicase activity using the substrate 3A (Tale S1) shown in A. Lane C is the reaction without enzyme and lane B is the heat-denatured substrate. Lanes 1–6 are reactions with increasing concentration of PfUDN. C, The structure of the linear substrate for the 3′ to 5′ direction (substrate 3B, Tale S1). An asterisk (*) denotes the 32P-labeled end. D, The helicase activity using the substrate 3B (Tale S1) shown in C. Lane C is the reaction without enzyme and lane B is the heat-denatured substrate. Lanes 1–6 are reactions with increasing concentration of PfUDN. E, The quantitative enzyme activity data from the autoradiogram in D are shown and the concentration of PfUDN used is also written.
Figure 9.
Immunoprecipitation and activity analysis of endogenous P. falciparum UvrD protein.
A. Western blot. Lane M is prestained marker. Lane 1 is immunoprecipitate using anti-PfUDN IgG, and lane 2 is immunoprecipitated sample using preimmune IgG. The IgGs were crosslinked to minimize the elution but little amount of heavy chain was detected in the western blot. PfUvrD protein band is marked with arrow. B. Helicase activity. The helicase assay was done using the normal substrate (substrate 1, Tale S1). Lanes 1–4, reactions with increasing concentration of elute using preimmune IgG and lanes 5–8, reactions with increasing concentration of elute using anti-PfUDN IgG. Lane B is boiled control and lane C is control without protein. C. The quantitative enzyme activity data from the autoradiogram in B are shown. The numbers correspond to the lanes in B. D. Nucleotide requirement of helicase activity of endogenous PfUvrD. Helicase activity of endogenous PfUvrD in the presence of lane 1, dCTP, lane 2, CTP, lane 3, dGTP, lane 4, GTP, lane 5, dATP, lane 6, ATP, lane 7, dTTP and lane 8, UTP. C1 and C2 are reactions without enzyme and B1 and B2 are heat denatured substrates respectively. Lane E is enzyme reaction of endogenous PfUvrD in the absence of any NTP or dNTP. E, The quantitative enzyme activity data from the autoradiogram in D are shown and the various NTPs/dNTPs used are also written.
Figure 10.
Protein-protein interaction study. A,
ELISA based protein-protein interaction study of PfMLH with PfUDN, PfUDC1, and PfUDC2. The proteins coated such as PfUDC1, PfUDC2, PfMLH and PfUDN are written in the first column followed by the interacting proteins. The interaction was done as reported in materials and methods section. This experiment was repeated at least three times and the quantitative data are shown. B, Western blots of protein-protein interaction. Lane 1 and 2 are eluted fractions of in vitro immunoprecipitates of anti PfUDN IgG protein A sepharose column, lane 3 is immunoprecipitate of preimmune IgG protein A sepharose column. Lane 1 is probed with anti-PfUDN antibody; lanes 2 and 3 were probed with anti-MLH antibody.
Figure 11.
Effect of PfMLH on the helicase activity.
A, Effect of PfMLH on the helicase activity of purified PfUDN using substrate 1 (Tale S1). Lane 1, 1 µl (10 nM) PfUDN, lane 2, 1 µl PfMLH (8 nM), lane 3, 1 µl PfUDN (10 nM) and 1 µl PfMLH (8 nM), lane 4, 1 µl PfUDN (10 nM) and 2 µl (16 nM) PfMLH, lane 5, 1 µl PfUDN (10 nM) and 3 µl PfMLH (24 nM), lane C, control reaction without any enzyme and lane B is the heat-denatured substrate. B and C, Effect of PfMLH on the helicase activity of purified PfUDN using direction-specific substrate (substrate 3B, Tale S1). Lane 1, 1 µl PfUDN (10 nM), lane 2, 1 µl PfUDN (10 nM) and 1 µl PfMLH (8 nM), lane 3, 1 µl PfUDN (10 nM) and 2 µl PfMLH (16 nM), lane 4, 1 µl PfUDN (10 nM) and 1 µl PfMLH (8 nM), lane 5, 1 µl PfUDN (10 nM) and 2 µl PfMLH (16 nM), lane C, control reaction without any enzyme. In lane 3 and 4, PfUDN and PfMLH were added at the same time in the substrate. In lane 5 and lane 6, the substrate mixture was pre-incubated with 1 and 2 µl of PfMLH for 10 minutes before the addition of PfUDN. The amount of unwound DNA was quantitated and plotted as a histogram above the gel picture. D, Effect of heat treated PfMLH, PfMLHN and PfMLHC on the helicase activity of purified PfUDN using direction-specific substrate (substrate 3B, Tale S1). Lane 1, 1.5 µl PfUDN (15 nM), lane 2, 1.5 µl PfUDN (15 nM) and 1 µl heat treated PfMLH (8 nM), lane 3, 1.5 µl PfUDN (15 nM) and 1 µl PfMLHN (8 nM), lane 4, 1.5 µl PfUDN (15 nM) and 1 µl PfMLHC (8 nM), lane 5, 1.5 µl PfUDN (15 nM) and 1 µl heat treated PfMLH (8 nM), lane 6, 1.5 µl PfUDN (15 nM) and 1 µl PfMLHN (8 nM), and lane 7, 1.5 µl PfUDN (15 nM) and 1 µl PfMLHC (8 nM). In lanes 2, 3 and 4, PfUDN and heat treated PfMLH, PfMLHN and PfMLHC were added at the same time in the substrate. In lane 5, 6 and 7, the substrate mixture was pre-incubated with 1 µl of heat treated PfMLH, PfMLHN and PfMLHC for 10 minutes before the addition of PfUDN. E, The amount of unwound DNA was quantitated and plotted as a histogram above the gel picture. F and G, Effect of PfUDN on the endonuclease activity of purified PfMLH. Lane 1, negative control reaction without enzyme, lane 2, reaction with purified PfMLH (20 nM), lane 3, reaction with purified PfMLH (20 nM) and PfUDN (15 nM) and lane 4, reaction with purified PfUDN (15 nM). H and I, Effect of PfUDC1 on the endonuclease activity of purified PfMLH. Lane 1, negative control reaction without enzyme, lane 2, reaction with purified PfMLH (20 nM), lane 3, reaction with purified PfMLH (20 nM) and PfUDC1 (15 nM) and lane 4, reaction with purified PfUDC1 (15 nM). The arrows show the position of nicked circular (N), linear (L) and supercoiled (S) plasmid DNA. The percent amount of nicked DNA was quantitated and plotted as a bar diagram above the gel picture. Each bar represents the mean percentage ± SD.
Figure 12.
A, Immunofluorescence staining. The cells were fixed and immunostained. Panel (i) phase (ii) image of cell stained with DAPI (iii) immunofluorescently stained cell (green, P. falciparum UvrD) (iv) immunofluorescently stained cell (red, P. falciparum MLH) and (v) super-imposed images. Control normal mouse sera produced no fluorescence (data not shown). B, Western blot analysis. Lane M is the protein molecular weight marker and lane 1 is protein from early schizont stages of the intraerythrocytic developmental of the parasite. The arrow shows the PfUvrD band.
Figure 13.
Models for PfMLH-stimulated unwinding. A,
Using normal partially duplex circular substrate (substrate 1, Tale S1). 1. Incubation with PfMLH. 2. It creates non-specific nicks. 3. It results in unstable substrate, which melts at 37°C and addition of PfUDN has no effect. B, Using direction-specific substrate (substrate 3B, Tale S1). 1. Incubation with PfMLH results in its binding to substrate. 2. Incubation with PfUDN facilitates its loading to the substrate through PfMLH. 3. It results in stimulation of unwinding in 3′ to 5′ direction.