Variable Inhibition of DNA Unwinding Rates Catalyzed by the SARS-CoV-2 Helicase Nsp13 by Structurally Distinct Single...

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• SARS-CoV-2 non-structural protein 13 (Nsp13) is a helicase crucial for viral replication.
• Nsp13 unwinds double-stranded RNA/DNA in the 5' to 3' direction.
• DNA lesions can impede viral replication by affecting helicase activity.

Purpose of the Study:

• To investigate the impact of distinct DNA lesions on SARS-CoV-2 Nsp13 DNA unwinding activity.
• To compare the effects of benzo[a]pyrene (B[a]P) adducts and UV-induced photolesions on helicase function.
• To determine whether unwinding rate or processivity is a better indicator of lesion inhibition.

Main Methods:

• Enzymatic assays measuring DNA unwinding kinetics.
• Quantification of unwinding rate constants (k_obs) and processivities (P).
• Analysis of Nsp13 activity on DNA containing B[a]P-dG adducts, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone (6-4PP) photolesions.

Main Results:

• DNA unwinding rate constants (k_obs) were reduced by up to 15-fold for B[a]P adducts and 2-5 fold for photolesions.
• Processivity (P) was reduced by 11% for a minor-groove B[a]P adduct and 67% for an intercalated adduct.
• Cyclobutane pyrimidine dimers (CPDs) caused the most significant reduction in processivity (approx. 90%), despite having a relatively high unwinding rate.

Conclusions:

• DNA unwinding by SARS-CoV-2 Nsp13 is significantly affected by the presence of DNA lesions.
• The inhibitory effect of DNA lesions on Nsp13 is lesion-dependent.
• Processivity is a more sensitive measure of the inhibitory effects of DNA lesions on helicase activity than unwinding rate constants.