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

The helicase may dissociate from the duplex DNA before it is completely unwound. Therefore, to investigate the processivity of nsP13, kinetic studies of nsP13-catalyzed DNA unwinding were performed with DNA substrates containing different duplex lengths (shown in Table 1 ) under the single-turnover reaction condition. Measurements of the reaction amplitude of the fraction of dsDNA molecules unwound allows one to calculate the processivity of the helicase (31, 32) . As shown in Figure 2 , the amplitude decreased as the length of the DNA duplex increased from 20 to 90 bp. This decrease in amplitude with an increase in duplex length may have occurred partly due to the reannealing of DNA strands behind the helicase and to the helicase dissociation from the DNA during unwinding. Nevertheless, the large excess of trap DNA that is included in the reaction rapidly anneals to the strand that is displaced from the duplex substrates during unwinding. A decrease in the amplitude of DNA unwound was observed in the absence of trap DNA at a given duplex DNA length (data not shown), indicating that the trap DNA prevented reannealing of displaced strands after completion of unwinding.