Inhibition of SARS-CoV-2 helicase by single DNA damage

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The SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 genome encodes several non-structural proteins (NSPs) involved in viral replication and propagation, with NSP13 known to play a role in RNA unfolding and transcription subsequent single stranded matrix.

To study: Variable inhibition of DNA unwinding rates catalyzed by SARS-Cov-2 helicase nsp13 (COV19) by structurally distinct unique DNA lesions. Image Credit: Limbitech / Shutterstock

NSP13 helicase can be inhibited by preventing interaction with target DNA. In an article recently uploaded to the Preprint Server bioRxiv*, the impact of specific DNA lesions is studied concerning the unwinding activity of NSP13.

How was the study carried out?

The 3 ‘end of an oligonucleotide substrate was labeled with a fluorescence quencher, while the 5’ end of the opposite strand was labeled with a fluorophore. Therefore, the fluorescence intensity was directly proportional to the activity of the applied NSP13 helicase, where dissociation of the oligonucleotide stops quenching of the fluorophore.

Specific DNA lesions were generated on the oligonucleotide substrate then identified and separated by nuclear magnetic resonance spectroscopy and high performance liquid chromatography. Two types of lesions studied were induced by exposure to UV light: cross-linked lesions of cyclobutane pyrimidine thymine dimer (CPD) and pyrimidine (6−4) pyrimidone (6-4PP) photoproduct, while two others were guanine adducts which produced either trans Where cis stereoisomers.

What did the study find?

All four types of DNA damage induced severe loss of unwinding activity by NSP13, with unwinding rates observed 20 to 100 times slower than unmodified DNA. In unmodified DNA, an initial explosion of unwinding activity occurs due to the rapid unwinding of preexisting NSP13-DNA duplexes, slowing down as subsequent complex formation becomes the limiting factor. Burst activity like this was not observed for DNA-bearing lesions. Thus, the group then investigated whether the lesions prevent the NSP13-DNA complex or only its subsequent activity.

TransDNA is characterized by an intact base pairing and an almost normal backbone, while cisThe DNA is significantly more distorted around the site of the lesion. As a result, NSP13 could interact better with transDNA, and significantly greater unwinding activity was observed. NSP13 forms a narrow channel through which the translocation strand must pass, and the additional steric hindrance generated by cisDNA was found to slow the unwinding speed. Likewise, DNA carrying the smaller CPD and 6-4PP lesions was found to unfold 2-5 times faster than the cis Where trans lesion.

The CPD lesion exhibits two crosslinked covalent bonds, while 6-4PP bears only one, but is overall more skeletal-deforming of DNA. Again, this directly results in a slower rate of unwinding by NSP13 for DNA carrying the 6-4PP lesion than CPD.

The authors note that the way DNA carrying the less bulky CPD or 6-4PP lesions or the larger guanine adducts unfolds differs not only in speed but also in character, with the speed changing over time. DNA bearing CPD or 6-4PP lesions was unwound by NSP13 in a nonlinear fashion until yields of 25-35% were achieved, at which time the rate of unwinding reached a plateau. Conversely, unwinding rates increased more slowly in a linear fashion over time for DNA carriers. cis Where trans guanine adducts at least 35% and potentially more.

*Important Notice

bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.

Journal reference:

  • Sales, A. et al. (2021) “Variable inhibition of DNA unwinding rates catalyzed by SARS-Cov-2 (COV19) nsp13 helicase by structurally distinct unique DNA lesions”. bioRxiv. do I: 10.1101 / 2021.10.13.464299.


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