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

The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' → 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[]pyrene (B[]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants () and processivities () were examined. Relative to undamaged DNA, the values were diminished by factors of up to ~15 for B[]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[]P adduct showed the smallest impact on , which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest value, exhibited the lowest , which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.