Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase (Science, 2007, 317:513-516)

報告日期: 2007/10/30
報告時間: 15:10/16:00
報告學生: 蕭光良
講評老師: 楊孔嘉
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Spring-Loaded Mechanism of DNA Unwinding by Hepatitis C Virus NS3 Helicase

Sua Myong, Michael M. Bruno, Anna M. Pyle, Taekjip Ha

Science 317: 513-516 (2007)

Speaker: 蕭光良

Commentator: 楊孔嘉博士

Date: 2007/10/30 15:10 - 16:00

Place: Lecture Room 602

Abstract

Nonstructural protein 3 (NS3) is an essential enzyme for hepatitis C virus replication. The NS3 protein is a bifunctional enzyme that contains both protease and helicase activities. Its helicase activity is very different from others: It can unwind both RNA and DNA substrates. In this study, the authors used single-molecular fluorescence resonance energy transfer (FRET) to resolve the elemental step of DNA separation catalyzed by NS3. The results showed, clearly, NS3 unwinding DNA in 3-bp steps. Dwell time analysis indicated that about three hidden steps are required in every 3-bp step, each hidden step linked to an adenosine triphosphate (ATP) hydrolysis. The helicase domain of NS3 has three domains. There is an ATP-binding pocket between domain 1 and domain 2. The structures, obtained in the presence and absence of ATP (or ATP analogs), of NS3 helicase domain and other structurally analogous proteins show that two highly conserved threonines, bind to two phosphates that are located 2 and 3 nt apart respectively. The domain 3 present a highly conserved aromatic residue that stacks against the base at the 3'end of duplex, which might keep the relative positions of domain 3 and DNA fixed while domains 1 and 2 translocate. The authors proposed the spring-loaded mechanism: Domain 1 and 2 of NS3 move along the tracking strand one nucleotide at a time in the 3' to 5' direction, consuming one ATP. The third domain lags behind by anchoring itself to the DNA in the first and second steps to accumulate tension. At the third step, the spring-loaded domain 3 moves forward in a burst motion, unzipping 3-bp as a consequence. The displaced strand is likely to be sheltered by the enzyme during unwinding. Upon encountering a barrier or after unwinding to 18bp, NS3 snaps or slips backward rapidly and repeats unwinding. Such repetitive unwinding behavior over a short stretch of duplex may help to keep the secondary structure of HCV genome melted during HCV replication.

Reference

Myong S, Bruno MM, Pyle AM, Ha T. Spring-Loaded Mechanism of DNA Unwinding by Hepatitis C Virus NS3 Helicase. Science 317: 513-516 (2007)