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The glutamate effect on DNA binding by DNA polymerases from Thermus aquaticus and Escherichia coli
Author(s) -
Deredge Daniel Joseph,
Thompson Gregory S,
Jiang Ke,
Patel Shree H,
Baker John T,
LiCata Vince J
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.591.3
Subject(s) - klenow fragment , dna polymerase i , thermus aquaticus , dna , chemistry , biochemistry , dna polymerase , biophysics , biology , polymerase chain reaction , gene , reverse transcriptase , exonuclease
DNA binding by Klenow ( E. coli ) and Klentaq ( T. aquaticus ) DNA polymerases has been characterized as a function of monovalent salt concentration, pH, and osmotic pressure. Nanomolar DNA binding by Klenow persists into much higher KCl concentration than Klentaq and results in the net release of 1–2 more ions. Both proteins exhibit a “glutamate effect,” wherein replacement of Cl− by glutamate (Glu−) as the primary anion results in a significant increase in DNA binding affinity. However, the linked ion release upon binding of Klentaq remains the same in Cl− versus Glu−, while it is reduced by >50% in Glu− for Klenow. Furthermore, the presence of glutamate also affected proton linkages of the two proteins differently. Salt displacement titrations suggest that Glu− is more excluded than Cl − from the surface of both proteins. Such exclusion could result in decreased electrostatic competition at low salt coupled with an enhanced osmotic effect at high salt which could explain the different character of the glutamate effects on the two proteins. Preliminary data suggests that the presence of Cl − versus Glu− can dramatically influence the osmotic stress effects on DNA binding by the polymerases. NSF.