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 investigate whether multiple nsP13s can track through the loading strand and participate in the unwinding of the DNA duplex, we prepared gapped DNA substrates with or without a 5 0 -ssDNA tail (Table 1) . Gapped substrates with a 5 0 -ssDNA tail, which contained a ssDNA gap that was 10 nt in length between the 30 bp of the lead duplex and 20 bp of the trailing duplex (20T30bp10G20bp in Table 1 ), was readily unwound by nsP13, resulting in displacement of the two duplexes ( Figure 5A ). However, for the gapped substrate that did not have the 5 0 -ssDNA tail (0T30bp10G20bp in Table 1 ), nsP13 was able to unwind only the 20 bp of the trailing duplex and not the 30 bp of the leading duplex ( Figure 5A ). This result indicates that the two gapped DNA substrates are recognized differently by nsP13 and the 5 0 -ssDNA loading strand is of primary importance for recognition and unwinding of the DNA duplex. Notably, a 10-nt gap was sufficient for the internal initiation site to completely unwind the 20 bp of the trailing DNA duplex, which was not observed for the unwinding of the longer 30-bp DNA duplex with a 10 nt gap (substrate '10 Gap' in Figure 4 ). This indicates that nsP13 requires the presence of a longer 5 0 -ssDNA track to unwind a longer duplex DNA.