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Effect of molecular crowding on DNA polymerase activity
Author(s) -
Sasaki Yoshiharu,
Miyoshi Daisuke,
Sugimoto Naoki
Publication year - 2006
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200500032
Subject(s) - polymerase , macromolecular crowding , in vivo , dna polymerase , peg ratio , polyethylene glycol , biophysics , in vitro , dna , chemistry , biochemistry , biology , microbiology and biotechnology , macromolecule , genetics , finance , economics
Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo . To understand biomolecular behavior in vivo , properties studied in vitro are extrapolated to conditions in vivo ; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro .