Cold stress-induced pea DNA helicase 47 is homologous to eIF4A and inhibited by DNA-interacting ligands.
Helicases are ubiquitous molecular motor proteins that play important role in maintaining the genome integrity and thus involved in plant growth and development. Here, we report the cloning of cDNA (1.64 kb) and genomic DNA (2.2 kb) of cold stress-induced pea DNA helicase 47 (PDH47) and characterization of its encoded protein. It belongs to DEAD-box protein family and shows striking identity (93%) with tobacco eIF4A. The transcript was induced under cold (4 degrees C) stress. The purified PDH47 protein (47 kDa) contains ATP-/Mg2+-dependent DNA unwinding as well as DNA-/Mg2+-dependent ATPase activities. The ATPase activity of PDH47 is stimulated more by ssDNA as compared to dsDNA and RNA. The activities of PDH47 are inhibited by various DNA-interacting ligands such as nogalamycin, daunorubicin, ethidium bromide, mitoxantrone, actinomycin, and cisplatin with apparent Ki values ranging from 0.5 to 8.0 microM. Interestingly, netropsin and distamycin inhibited the helicase but not the ATPase activity. The inhibition might be due to the intercalation of inhibitors into duplex DNA, which can impede the translocation of the PDH47. This study should help in our better understanding of cold stress signaling and mechanism of DNA unwinding in plants.
Vashisht
Ajay Amar
AA
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Tuteja
Narendra
N
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A / Q000737:chemistry*; D007364:Intercalating Agents / Q000494:pharmacology*; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D018532:Pisum sativum / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; D007364:Intercalating Agents; D008024:Ligands; D019384:Nucleic Acid Synthesis Inhibitors; D000255:Adenosine Triphosphate; D004247:DNA; D039601:Eukaryotic Initiation Factor-4A; D017301:Ca(2+) Mg(2+)-ATPase; D004265:DNA Helicases