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Berezhna SY Gill JP Lamichhane R Millar DP 《Journal of the American Chemical Society》2012,134(27):11261-11268
Enzymatic reactions typically involve complex dynamics during substrate binding, conformational rearrangement, chemistry, and product release. The noncovalent steps provide kinetic checkpoints that contribute to the overall specificity of enzymatic reactions. DNA polymerases perform DNA replication with outstanding fidelity by actively rejecting noncognate nucleotide substrates early in the reaction pathway. Substrates are delivered to the active site by a flexible fingers subdomain of the enzyme, as it converts from an open to a closed conformation. The conformational dynamics of the fingers subdomain might also play a role in nucleotide selection, although the precise role is currently unknown. Using single-molecule F?rster resonance energy transfer, we observed individual Escherichia coli DNA polymerase I (Klenow fragment) molecules performing substrate selection. We discovered that the fingers subdomain actually samples through three distinct conformations--open, closed, and a previously unrecognized intermediate conformation. We measured the overall dissociation rate of the polymerase-DNA complex and the distribution among the various conformational states in the absence and presence of nucleotide substrates, which were either correct or incorrect. Correct substrates promote rapid progression of the polymerase to the catalytically competent closed conformation, whereas incorrect nucleotides block the enzyme in the intermediate conformation and induce rapid dissociation from DNA. Remarkably, incorrect nucleotide substrates also promote partitioning of DNA to the spatially separated 3'-5' exonuclease domain, providing an additional mechanism to prevent misincorporation at the polymerase active site. These results reveal the existence of an early innate fidelity checkpoint, rejecting incorrect nucleotide substrates before the enzyme encloses the nascent base pair. 相似文献
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Zegers RG van Den Berg AM Brandenburg S Fleurot FR Fujiwara M Guillot J Hannen VM Harakeh MN Laurent H van Der Schaaf K van Der Werf SY Willis A Wilschut HW 《Physical review letters》2000,84(17):3779-3782
The (nat)Pb(3He,tp) reaction at E(3He) = 177 MeV was studied to identify 2Planck's over 2piomega isovector monopole strength in Bi isotopes. Monopole strength was found in the region -45
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The dynamics of a single strain HIV model is studied.The basic reproduction number R_0 used as a bifurcation parameter shows that the system undergoes transcritical and saddle-node bifurcations.The usual threshold unit value of R_0 does not completely determine the eradication of the disease in an HIV infected person.In particular,a sub-threshold value R_C is established which determines the system's number of endemic states:multiple if R_c Ro 1,only one if R_C = R_0 = 1,and none if R_0 R_C 1. 相似文献
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