DOI QR코드

DOI QR Code

T7 박테리오파지 gp4 DNA helicase에 의한 DNA unwinding에서 step size의 반응속도론적 측정

Kinetic Measurement of the Step Size of DNA Unwinding by Bacteriophage T7 DNA Helicase gp4

  • Kim, Dong-Eun (Department of Biotechnology and Bioengineering, Dong-Eui University)
  • 발행 : 2004.02.01

초록

T7박테리오파지 gp4는 dTTP 가수분해에너지를 이용하여 DNA복제시 이중 나선 DNA를 단일가닥 DNA로 풀어내는 나선효소(helicase)이다. T7 나선효소의 활성형의 4차구조는 한가운데 구멍을 지닌 육량체 고리모양이다. 단일가닥 DNA는 나선효소가 $5'\rightarrow3'$방향으로 이동할 때 육량체 고리의 구멍으로 빠져나간다. 이러한 DNA의 이중나선 풀어헤침을 빠른 효소반응속도 측정법을 이용하여 정량적으로 측정하였으며, 그 결과 단일가닥 DNA 산물들이 생성되기 전에 지연상태(lag phase)가 존재함을 관찰하였다. 이러한 지연상태를 나선효소에 의한 이중나선 DNA의 풀어헤침이 속도론적 단계과정(kinetic stepping)을 거친다는 모델로써 분석하였다. 예상대로 이중나선의 길이가 클수록 지연상태의 지속시간이 늘어났다. $\tau7$ 나선효소가 이중나선 DNA를 풀어내는 과정에서 넣어준 trap DNA는 풀어내는 이중나선 DNA의 양을 변화시키지 못하여서, $\tau7$ 나선효소가 매우 큰 공정성을 지닌 효소임을 알 수 있었다. 이러한 속도론적 data를 global fitting법을 써서 kinetic stepping 모델에 적용한 결과 매 단계(step)마다 10∼l개의 염기쌍이 풀려지고 1초당 3.7번의 step이 일어난다는 것을 알 수 있었다. DNA 풀어헤침과 dTTP가수분해의 메커니즘과 이들의 연계성은 $4∼37^{\circ}C$사이의 온도범위에서 영향을 받지 않았다. 이상을 종합할 때, T7나선효소의 이중나선 DNA의 풀어헤침 시 나타나는 속도론적 단계과정은 DNA복제 시 이용되는 나선효소의 내재적 속성임을 알 수 있다.

T7 bacteriophage gp4 is the replicative DNA helicase that unwinds double-stranded DNA by utilizing dTTP hydrolysis energy. The quaternary structure of the active form of T7 helicase is a hexameric ring with a central channel. Single-stranded DNA passes through the central channel of the hexameric ring as the helicase translocates $5'\rightarrow3'$ along the single-stranded DNA. The DNA unwinding was measured by rapid kinetic methods and showed a lag before the single-stranded DNA started to accumulate exponentially. This behavior was analyzed by a kinetic stepping model for the unwinding process. The observed lag phase increased as predicted by the model with increasing double-stranded DNA length. Trap DNA added in the reaction had no effect on the amplitudes of double-stranded DNA unwound, indicating that the $\tau7$ helicase is a highly processive helicase. Global fitting of the kinetic data to the stepping model provided a kinetic step size of 10-11 bp/step with a rate of $3.7 s^{-1}$ per step. Both the mechanism of DNA unwinding and dTTP hydrolysis and the coupling between the two are unaffected by temperature from $4∼37^{\circ}C$. Thus, the kinetic stepping for dsDNA unwinding is an inherent property of tile replicative DNA helicase.

키워드

참고문헌

  1. J. Biol. Chem. v.272 Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'-and 3'-tails of the forked DNA substrate Ahnert,P.;Patel,S.S. https://doi.org/10.1074/jbc.272.51.32267
  2. EMBO J. v.19 A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein Ahnert,P.;Picha,K.M.;Patel,S.S. https://doi.org/10.1093/emboj/19.13.3418
  3. Science v.275 Kinetic measurement of the step size of DNA unwinding by Escherichia coil UvrD helicase Ali,J.A.;Lohman,T.M. https://doi.org/10.1126/science.275.5298.377
  4. J. Mol. Biol. v.293 An oligomeric form of E. coli UvrD is required for optimal helicase activity Ali,J.A.;Maluf,N.K.;Lohman,T.M. https://doi.org/10.1006/jmbi.1999.3185
  5. J. Biol. Chem. v.256 Rep protein as a helicase in an active, isolatable repolication fork of duplex phi X174 DNA Arai,N.;Kornberg,A.
  6. J. Biol. Chem. v.262 Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro Baker,T.A.;Funnell,B.E.;Kornberg,A.
  7. Biochemistry v.33 Single-turnover kinetics of helicase-catalyzed DNA unwinding monitored continuously by fluoresence energy transfer Bjornson,K.P.;Amaratunga,M.;Moore,K.J.;Lohman,T.M. https://doi.org/10.1021/bi00251a044
  8. Biochemistry v.39 Domonstration of unidirectional single-stranded DNA translocation by PcrA helicase: measurement of step size and translocation speed Dillingham,M.S.;Wigley,D.B.;Webb,M.R. https://doi.org/10.1021/bi992105o
  9. Proc. Natl. Acad. Sci. USA v.92 Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases Englman,E.H.;Yu,X.;Wild,R.;Hingorani,M.M.;Patel,S.S. https://doi.org/10.1073/pnas.92.9.3869
  10. Kintics for the Life Sciences. Receptors, Transmitters and Catalysts Gutfreund,H.
  11. Biochemistry v.36 A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding Hacker,K.J.;Johnson,K.A. https://doi.org/10.1021/bi971644v
  12. Biochemistry v.35 Cooperative interactions of nucleotide ligands are linked to oligomerization and DNA binding in bacteriophage T7 gene 4 helicases Hingorani,M.M.;Patel,S.S. https://doi.org/10.1021/bi9521497
  13. Nature v.403 The DExH protein NPH-Ⅱ is a processive and directional motor for unwinding RNA Jankowsky,E.;Gross,C.H.;Shuman,S.;Pyle,A.M. https://doi.org/10.1038/35000239
  14. J. Mol. Biol. v.321 T7 DNA helicase: a molecular motor that processively and unidirectionally translocates along single-stranded DNA Kim,D.E.;Narayan,M.;Patel,S.S. https://doi.org/10.1016/S0022-2836(02)00733-7
  15. Biochemistry v.32 Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli Korangy,F.;Julin,D.A. https://doi.org/10.1021/bi00069a024
  16. Biochemistry v.33 Efficiency of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli Korangy,F.;Julin,D.A. https://doi.org/10.1021/bi00198a022
  17. J. Biol. Chem. v.261 The Escherichia coli dnaB replication protein in a DNA helicase LeBowitz,J.H.;McMacken,R.
  18. Annu. Rev. Biochem. v.65 Mechanisms of helicase-catalyzed DNA unwinding Lohman,T.M.;Bjornson,K.P. https://doi.org/10.1146/annurev.bi.65.070196.001125
  19. Annu. Rev. biochem. v.69 Structure and Function of Hexameric Helicases Patel,S.S.;Picha,K.M. https://doi.org/10.1146/annurev.biochem.69.1.651
  20. J. Biol. Chem. v.267 Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation Patel,S.S.;Rosenberg,A.H.;Studier,F.W.;Johnson,K.A.
  21. J. Biol. Chem. v.273 Bacteriophage T7 DNA helicase binds dTTP forms hexamers, and binds DNA in the absence of Mg$^{2+}$. The presence of dTTP is sufficient for hexamer formation and DNA binding Picha,K.M.;Patel,S.S. https://doi.org/10.1074/jbc.273.42.27315
  22. J. Biol. Chem. v.267 Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme Roman,L.J.;Eggleston,A.K.;Kowalczykowski,S.C.
  23. Biochemistry v.28 Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay Roman,L.J.;Kowalczykowski,S.C. https://doi.org/10.1021/bi00433a018
  24. Biochemistry v.28 Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA Roman,L.J.;Kowalczykowski,S.C. https://doi.org/10.1021/bi00433a019
  25. Molecular Cloning, A Laboratory Manual(second edn.) Single stranded, Filamentous Bacteriophage Vectors Sambrook,J.;Fritsch,E.F.;Maniatis,T.;Ford,N.(ed.);Nolan,C.(ed.);Ferguson,M.(ed.)
  26. Biochemistry v.36 Kinetics of the RNA-DNA helicase activity of Escherichia coli transcription termination factor rho. 2. Processivity, ATP consumption, and RNA binding Walstrom,K.M.;Dozono,J.M.;von Hippel, P.H. https://doi.org/10.1021/bi963180r
  27. Nature Struct. Biol. v.3 DNA is bound within the central hole to one or two of the six subunits of the T7 DNA helicase Yu,X.;Hingorani,M.M.;Patel,S.S.;Egelman,E.H. https://doi.org/10.1038/nsb0996-740