ATPase-dependent duplex nucleic acid unwinding by SARS-CoV-2 nsP13 relies on facile binding and translocation along single-stranded nucleic acid

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 13 (nsP13) is a superfamily 1 helicase crucial for viral RNA replication.
• nsP13 unwinds double-stranded RNA and DNA with a 5' single-stranded tail in a 5'-3' direction.
• The precise mechanism of nsP13 unwinding, despite its known dependence on ATP hydrolysis, remains incompletely understood.

Purpose of the Study:

• To investigate the mechanism of nucleic acid unwinding by SARS-CoV-2 nsP13.
• To determine if ATP hydrolysis-induced changes in nsP13 binding affinity to single-stranded tails influence unwinding efficiency.

Main Methods:

• Investigated the unwinding of double-stranded DNA (dsDNA) with a 5' single-stranded tail by nsP13.
• Utilized ATP and a slowly hydrolyzable ATP analog, adenosine-5'-O-3-thiotriphosphate (ATPγS), to assess the role of ATP hydrolysis.
• Measured changes in nsP13 binding affinity to single-stranded DNA (ssDNA).

Main Results:

• A longer 5' single-stranded tail enhanced ATP hydrolysis and improved dsDNA unwinding efficiency by nsP13.
• Using ATPγS significantly diminished dsDNA unwinding by nsP13 compared to ATP.
• ATPγS resulted in enhanced binding affinity of nsP13 to ssDNA compared to ATP.

Conclusions:

• Unhindered ATP hydrolysis by nsP13 is critical for efficient binding and translocation along single-stranded nucleic acids.
• This process facilitates the effective unwinding of duplex nucleic acids.
• The study elucidates a key aspect of the nsP13 helicase mechanism essential for SARS-CoV-2 replication.