Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13

Lee, Na-Ra; Kwon, Hyun-Mi; Park, Kkothanahreum; Oh, Sangtaek; Jeong, Yong-Joo; Kim, Dong-Eun

Because nsP13 has been shown to unwind both DNA and RNA duplexes that have a 5 0 -tail (6,7), we designed DNA duplexes with a 5 0 -tail at one side as the unwinding substrates to investigate the single-turnover kinetics of DNA unwinding by the helicase. Previously, analysis of nucleic acids unwinding by the coronavirus helicase was performed using a multiple turnover kinetics experiment, in which the helicase may rebind to the substrates after completion of a cycle of unwinding (6, 7) . In contrast, our experimental setup of the single-turnover condition enabled us to obtain quantitative aspects of the unwinding kinetics, especially the processivity of unwinding by the helicase. The reaction scheme is shown in Figure 1A , in which the helicase is initially allowed to bind to the substrate without ATP hydrolysis. In the absence of ATP hydrolysis, unwinding was not observed (data not shown). However, the addition of magnesium ions to the reaction allowed the helicase to unwind the DNA duplexes ( Figure 1A ). The duplex DNA unwinding reaction was initiated by adding a solution of MgCl 2 (13 mM) and a large excess (3.0 mM) of trap oligonucleotides (unlabeled bottom strand) to a mixture of the pre-incubated reaction containing duplex DNA substrates (5 nM) and helicase (200 nM). The trap oligonucleotides served to prevent the re-initiation of unwinding by trapping free helicases and