ATPase Activity of Escherichia coli Rep Helicase Is Dramatically Dependent on DNA Ligation and Protein Oligomeric States
详细信息 查看全文
- 作者：Isaac Wong ; Keith J. M. Moore ; Keith P. Bjornson ; John Hsieh ; and Timothy M. Lohman
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：May 7, 1996
- 年：1996
- 卷：35
- 期：18
- 页码：5726 - 5734
- 全文大小：491K
- 年卷期：v.35,no.18(May 7, 1996)
- ISSN：1520-4995
文摘
The Escherichia coli Rep helicase catalyzes theunwinding of duplex DNA using the energyderived from ATP binding and hydrolysis. Rep functions as a dimerbut assembles to its active dimericform only on binding DNA. Each protomer of a dimer contains a DNAbinding site that can bind eithersingle-stranded (S) or duplex (D) DNA. The dimer can bind up totwo oligodeoxynucleotides in fiveDNA-ligation states: two half-ligated states, P2S andP2D, and three fully-ligated states,P2S2, P2D2,andP2SD. We have previously shown that the relativestabilities of these ligation states are allostericallyregulated by the binding and hydrolysis of ATP and have proposed an"active rolling" model for DNAunwinding where the enzyme cycles through a series of these ligationstates in a process that is coupledto the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992) Science 256,350-355].The basal ATPase activity of Rep protein is stimulated by ss DNAbinding and by protein dimerization.We have measured the steady-state ATPase activities of Rep boundto dT(pT)15 in each distinct ss DNAligation state (PS, P2S, and P2S2)to compare with our previous measurements with unligated Repmonomer(P) [Moore, K. J. M., & Lohman, T. M. (1994) Biochemistry33, 14550]. We find the ATPaseactivityof Rep is influenced dramatically by both dimerization and ss DNAligation state, with the followingkcatvalues for ATP hydrolysis increasing by over 4 orders of magnitude:2.1 × 10-3 s-1for P, 2.17 ± 0.04s-1 for PS, 16.5 ± 0.2s-1 for P2S, and 71 ± 2.5s-1 for P2S2 (20 mMTris-HCl, pH 7.5, 6 mM NaCl, 5mM MgCl2, 10% glycerol, 4 
C). The apparentKM's for ATP hydrolysis are 2.05 ± 0.1 
Mfor PS and2.7 ± 0.2 M for P2S. These widely differentATPase activities reflect the allosteric effects of DNAligation and demonstrate that cooperative communication occurs betweenthe ATP and DNA sites ofboth subunits of the Rep dimer. These results further emphasizethe need to explicitly consider thepopulation distribution of oligomerization and DNA ligation states ofthe helicase when attempting toinfer information about elementary processes such as helicasetranslocation based solely on macroscopicsteady-state ATPase measurements.
C). The apparentKM's for ATP hydrolysis are 2.05 ± 0.1 Mfor PS and2.7 ± 0.2 M for P2S. These widely differentATPase activities reflect the allosteric effects of DNAligation and demonstrate that cooperative communication occurs betweenthe ATP and DNA sites ofboth subunits of the Rep dimer. These results further emphasizethe need to explicitly consider thepopulation distribution of oligomerization and DNA ligation states ofthe helicase when attempting toinfer information about elementary processes such as helicasetranslocation based solely on macroscopicsteady-state ATPase measurements.