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

It was of interest to determine whether nsP13 might traverse from the 5 0 -ssDNA tail to the internal gap and facilitate unwinding of the incoming duplex DNA, like a train on a railroad track pushing another train in the lead. To this end, we prepared DNA substrates with a longer trailing duplex (30 bp) and 10-nt gap (substrates in Figure 5B ). As stated earlier, the 10-nt gap was not sufficient for nsP13 to completely unwind the 30-bp DNA duplex on the 3 0 -side, which was unwound when the gapped DNA substrate without the 5 0 -ssDNA overhang was used (0T20bp10G30bp in Figure 5B ). Note that a blunt-ended DNA substrate (the lead 20-bp duplex of 0T20bp10G30bp) slightly unwound under this experimental setup. A portion of the blunt end duplex might have a 5 0 -ssDNA at the end due to breathing of the DNA duplex, which supports unwinding of the 20-bp DNA duplex. However, this effect was not sufficient for the unwinding of the longer 30-bp DNA duplex (square symbols in Figure 5A ). Most importantly, nsP13 was able to completely unwind the 30 bp of the trailing duplex in the 10-nt gapped DNA substrate containing a 5 0 -ssDNA tail (20T20bp10G30bp in Figure 5B ). This result suggests that the translocated nsP13 molecules pile up and facilitate unwinding of the 3 0 -side of the DNA duplex by the helicases that are internally bound at the gap. This may be the reason why the 30-bp DNA duplex containing the 10-nt gap and 5 0 -overhang (20T20bp10G30bp) unwound more efficiently than the gapped DNA substrate containing the longer lead DNA duplex (30 bp) with a blunt end ('10 Gap' shown in Figure 4) . A portion of the nsP13 helicases may stack up after the 5 0 -side of the DNA duplex is unwound and may then unwind the 3 0 -side of the DNA duplex. Therefore, the nsP13 helicase can track through the loading strand and facilitate unwinding of the DNA duplex as long as the translocated helicases stay on the track.