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

Time-courses of duplex DNA unwinding by 100 nM nsP13 with 2.5 nM of each duplex DNA substrate with various 5 0 -tail lengths (0, 5, 10, 15, 20, 30 and 40 nt) are shown in Figure 3 . The quantity of unwound duplex DNA increased as the length of the ss 5 0 -tail increased. These results suggest that more ssDNA products were formed from duplex DNA substrates that could potentially bind more than one helicase molecule on the 5 0 -tail, implying that multiple nsP13 molecules might exhibit a higher processivity. Alternatively, a tighter binding affinity of the helicase monomer or oligomers to longer ss tails might also explain this increase in DNA unwinding. Based on these two explanations, we hypothesized that the helicase may occupy the defined length of the ssDNA overhang as a minimal binding site size and the stronger affinity is caused by cooperative binding of multiple helicases on a longer ssDNA. A tail length of 20 nt enabled nsP13 to marginally unwind 50 bp duplex DNA substrates, and a tail length of 40 nt allowed nsP13 to maximally unwind more duplex DNA (>95%) that was 50 bp in length. We postulate that the functional nsP13 monomer or oligomer requires a ssDNA loading strand that is at least 20 nt to translocate and unwind duplex DNA, which is analogous to a putative binding site size.