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

When blunt ended duplex DNA with an internal gap of 0 nt (nicked DNA) was reacted with the nsP13 helicase, no ssDNA product was observed, suggesting that the presence of ssDNA of a certain length is required for unwinding by nsP13. Increasing the gap size allowed nsP13 to bind to ssDNA and to unwind the bottom strand DNA annealed to the 3 0 side of the top strand. When gaps of 5 or 10 nt were present in the bottom strand, the nsP13 helicase unwound the substrate with a very low processivity; <10% of substrates were unwound ( Figure 4B ). However, more ssDNA was formed when substrates contained gaps of 15 or 20 nt in the middle of the duplex DNA ( Figure 4B ). This result indicates that the longer loading strand facilitated more binding of nsP13 molecules to the ssDNA gap, resulting in a higher processivity in duplex (30 bp) unwinding. An increase in the amplitude of ssDNA product formation for DNA substrates containing 5-20 nt gaps was observed, indicating that DNA unwinding processivity was enhanced by an increase in the ssDNA gap. Interestingly, the amplitude obtained for 30-bp duplex DNA substrates containing a 20-nt gap was not as high as when the substrate contained a 20-nt 5 0 -tail overhang (>0.9 in Figure 2 ). This result suggests that the state of the nsP13 helicases bound to the 20-nt ssDNA gap were different from the one bound to the 20-nt 5 0 -tail.