Author: Lee, Na-Ra; Kwon, Hyun-Mi; Park, Kkothanahreum; Oh, Sangtaek; Jeong, Yong-Joo; Kim, Dong-Eun
Title: Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13 Document date: 2010_7_29
ID: 1k99yv4i_8
Snippet: The nsP13 (200 nM) and 32 P -labeled DNA substrates (5 nM) were mixed in buffer MixA [50 mM Tris-Cl (pH 6.8), 50 mM NaCl, 2 mM ATP, 5 mM EDTA and 10% glycerol]. The mixture was preincubated for 5 min at 22 C and the unwinding reactions were initiated by adding an equal volume of MixB [2 mM ATP, 13 mM MgCl 2 and 3 mM trap DNA (unlabeled bottom strand)]. After various times, the reactions were quenched by adding an equal volume of the quenching sol.....
Document: The nsP13 (200 nM) and 32 P -labeled DNA substrates (5 nM) were mixed in buffer MixA [50 mM Tris-Cl (pH 6.8), 50 mM NaCl, 2 mM ATP, 5 mM EDTA and 10% glycerol]. The mixture was preincubated for 5 min at 22 C and the unwinding reactions were initiated by adding an equal volume of MixB [2 mM ATP, 13 mM MgCl 2 and 3 mM trap DNA (unlabeled bottom strand)]. After various times, the reactions were quenched by adding an equal volume of the quenching solution (100 mM EDTA, 0.4% SDS, 20% glycerol and 0.1% bromophenol blue). The ds and ssDNAs were resolved by native (urea-free) PAGE. Size markers for the unwound products were produced by heating the duplex substrates with 600-fold trap DNA at 95 C. The radioactivity was quantified using a Cyclone (PerkinElmer) and analyzed by OptiQuant/Cyclone software (Packard Instrument Company). The ratio of unwound products was calculated as described earlier (27) and the data were fit to a single-exponential equation [Equation (1) ].
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