A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3'-->5' 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 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'-->5' 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 5.6, 5.2, 4.0 and 0.71 micro 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.
Phan
Tuan-Nghia
TN
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Ehtesham
Nasreen Z
NZ
Tuteja
Renu
R
Tuteja
Narendra
N
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology*; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D003630:Daunorubicin / Q000494:pharmacology; D019281:Dimerization; D004996:Ethidium / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009621:Nogalamycin / Q000494:pharmacology; D018532:Pisum sativum / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; C030013:cactinomycin; D003609:Dactinomycin; D004265:DNA Helicases; D004996:Ethidium; D009621:Nogalamycin; D003630:Daunorubicin