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The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C virus Helicase NS3h is Dependent on Both the Sugar and Base Moieties
Khaki, Ali R. Field, Cassandra Malik, Shuja Shafi Niedziela-Majka, Anita Leavitt, Stephanie A. Wang, Ruth Hung, Magdeleine Sakowicz, Roman Brendza, Katherine M. Fischer, Christopher J.
Khaki, Ali R.
Field, Cassandra
Malik, Shuja Shafi
Niedziela-Majka, Anita
Leavitt, Stephanie A.
Wang, Ruth
Hung, Magdeleine
Sakowicz, Roman
Brendza, Katherine M.
Fischer, Christopher J.
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Abstract
The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.
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Date
2010-05-06
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Elsevier
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Keywords
NS3 helicase, Translocation, Nucleic acid, ATPase, Mechanism
Citation
Khaki, Ali R., Cassandra Field, Shuja Malik, Anita Niedziela-Majka, Stephanie A. Leavitt, Ruth Wang, Magdeleine Hung, Roman Sakowicz, Katherine M. Brendza, and Christopher J. Fischer. "The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C Virus Helicase NS3h Is Dependent on Both Sugar and Base Moieties." Journal of Molecular Biology 400.3 (2010): 354-78.