Plasmodium falciparum UvrD Helicase Translocates in 3′ to 5′ Direction, Colocalizes with MLH and Modulates Its Activity through Physical Interaction
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.