TY - JOUR T1 - Mechanism of duplex unwinding by coronavirus nsp13 helicases JF - bioRxiv DO - 10.1101/2020.08.02.233510 SP - 2020.08.02.233510 AU - Xiao Hu AU - Wei Hao AU - Bo Qin AU - Zhiqi Tian AU - Ziheng Li AU - Pengjiao Hou AU - Rong Zhao AU - Sheng Cui AU - Jiajie Diao Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/08/03/2020.08.02.233510.abstract N2 - The current COVID-19 pandemic urges in-depth investigation into proteins encoded with coronavirus (CoV), especially conserved CoV replicases. The nsp13 of highly pathogenic MERS-CoV, SARS-CoV-2, and SARS-CoV exhibit the most conserved CoV replicases. Using single-molecule FRET, we observed that MERS-CoV nsp13 unwound DNA in discrete steps of approximately 9 bp when ATP was used. If another NTP was used, then the steps were only 4 to 5 bp. In dwell time analysis, we detected 3 or 4 hidden steps in each unwinding process, which indicated the hydrolysis of 3 or 4 dTTP. Based on crystallographic and biochemical studies of CoV nsp13 helicases, we modeled an unwinding mechanism similar to the spring-loaded mechanism of HCV NS3 helicase, although our model proposes that flexible 1B and stalk domains, by allowing a lag greater than 4 bp during unwinding, cause the accumulated tension on the nsp13-DNA complex. The hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible than in MERS-CoV nsp13 due to the difference of a single amino acid, which causes the former to induce significantly greater NTP hydrolysis. Our findings thus establish a blueprint for determining the unwinding mechanism of a unique helicase family.When dTTP was used as the energy source, 4 hidden steps in each individual unwinding step after 3 - 4 NTP hydrolysis were observed.An unwinding model of MERS-CoV-nsp13 which is similar to the spring-loaded mechanism of HCV NS3 helicase, except the accumulation of tension on nsp13/DNA complex is caused by the flexible 1B and stalk domains that allow a lag of 4-bp in unwinding.Comparing to MERS-CoV nsp13, the hinge region between two RecA-like domains in SARS-CoV-2 nsp13 is intrinsically more flexible due to a single amino acid difference, which contributes to the significantly higher NTP hydrolysis by SARS-CoV-2 nsp13.Competing Interest StatementThe authors have declared no competing interest. ER -