tailieunhanh - Báo cáo khoa học: A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3¢fi5¢ direction

A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chroma-tography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). | Eur. J. Biochem. 270 1735-1745 2003 FEBS 2003 doi A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3 fi5 direction Tuan-Nghia Phan Nasreen Z. Ehtesham Renu Tuteja and Narendra Tuteja International Centre for Genetic Engineering and Biotechnology New Delhi India A novel ATP-dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 PDH120 . The enzyme can unwind 17-bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3 fi5 direction. The enzyme also exhibits intrinsic single-stranded DNA- and Mg2 -dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency ATP GTP dCTP UTP dTTP CTP dATP dGTP for which divalent cation Mg2 Mn2 is required. The DNA intercalating agents actinomycin C1 ethidium bromide daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of and Ms respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA which results in the formation of DNA-inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants. Keywords DNA-dependent ATPase helicase inhibitors plant DNA helicase unwinding enzyme. Despite the energetically stable genomes of all living organisms including plants they have to