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

To further examine the effect of the length of the loading stand on nsP13 DNA duplex unwinding kinetics, we designed a series of duplex DNA substrates containing a DNA gap on the bottom strand (Table 1 and Figure 4 ). The gapped DNA duplex substrates did not have a 5 0 -tail at either end, which is available as loading strand for the helicase. Instead, the nsP13 helicase may recognize the internal ssDNA portion in the gapped DNA as the loading strand. The gap size was varied from 0 to 20 nt and unwinding at the bottom of the DNA strand (30 nt) annealed to the 3 0 -side of the top strand was monitored. Because entry of the helicase is not available at both blunt ends of the gapped DNA, the only available entry point of the helicase would be the ssDNA gap. In these experiments, the bottom strand (30 nt) annealed to the 5 0 -side of the top strand was displaced only when an overhang was present at the 5 0 -end of the gapped DNA duplex (data not shown).