Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13

Abstract

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5′-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5′-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5′-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5′-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5′-overhang account for the observed enhanced processivity of DNA unwinding.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…The protein is able to unwind dsRNA and dsDNA in a 5′-to-3′ direction with the energy obtained by the hydrolysis of all NTPs and dNTPs (136–140). It was proposed that the resulting ssRNAs probably serve as templates for RNA synthesis by the RdRp.…”

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

“…A helicase domain of superfamily 1 helicases is harbored in SARS-CoV nsp13 C-terminal half (Ivanov et al, 2004b). SARS-CoV nsp13 is able to unwind dsRNA (and dsDNA) in a 5 -3 direction and required energy obtained from the hydrolysis of any NTPs and dNTPs (Ivanov et al, 2004b;Lee et al, 2010;Tanner et al, 2003). The N-terminal region of nsp13 contains a zinc-binding domain forms of 12 conserved cysteine and histidine residues.…”

Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13

“…In order to accomplish this goal, we prepared natural chemical stocks (Table 1) and examined their effects on the activity of SARS-CoV helicase, nsP13. 6 We first attempted to screen compounds that suppress the DNA unwinding activity of nsP13 and the dsDNA unwinding activity of nsP13 was measured using a fluorometric assay, based on the FRET from the fluorescein to the carboxytetramethylrhodamine (TAMRA) (Fig. 6 We first attempted to screen compounds that suppress the DNA unwinding activity of nsP13 and the dsDNA unwinding activity of nsP13 was measured using a fluorometric assay, based on the FRET from the fluorescein to the carboxytetramethylrhodamine (TAMRA) (Fig.…”