The macroscopic rate of nucleic acid translocation by hepatitis C virus helicase NS3h is dependent on both sugar and base moieties.
The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s(-1) (oligo(dT)), 35 nt s(-1) (oligo(dU)), and 42 nt s(-1) (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K(0.5) for NS3h translocation at a saturating ATP concentration are as follows: 3.3+/-0.4 microM nucleotide (poly(dT)), 27+/-2 microM nucleotide (poly(dU)), and 36+/-2 microM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TDeltaS(0) are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (approximately 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.
Khaki
Ali R
AR
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA.
Field
Cassandra
C
Malik
Shuja
S
Niedziela-Majka
Anita
A
Leavitt
Stephanie A
SA
Wang
Ruth
R
Hung
Magdeleine
M
Sakowicz
Roman
R
Brendza
Katherine M
KM
Fischer
Christopher J
CJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism*; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D009711:Nucleotides; D012313:RNA; D020365:RNA Helicases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
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