A pre-equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1.
J Biol Chem
; 282(39): 28884-28892, 2007 Sep 28.
Article
em En
| MEDLINE
| ID: mdl-17640877
ABSTRACT
Numerous studies have been undertaken to establish the mechanism of dNTP binding and template-directed incorporation by DNA polymerases. It has been established by kinetic experiments that a rate-limiting step, crucial for dNTP selection, occurs before chemical bond formation. Crystallographic studies indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. In previous studies, we established a fluorescence resonance energy transfer system to monitor the open-to-closed transition in the fingers subdomain of Klentaq1. By comparing the rates of the fingers subdomain closure with that of the rate-limiting step for Klentaq1, we showed that fingers subdomain motion was significantly faster than the rate-limiting step. We have now used this system to characterize DNA binding as well as to complete a more extensive characterization of incorporation of all four dNTPs. The data indicate that DNA binding occurs by a two-step association and that dissociation of the DNA is significantly slower in the case of the closed ternary complex. The data for nucleotide incorporation indicate a step occurring before dNTP binding, which differs for all four nucleotides. As the only difference between the (E x p/t) complexes is the templating base, it would suggest an important role for the templating base in initial ground state selection.
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Base de dados:
MEDLINE
Assunto principal:
DNA
/
Modelos Moleculares
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DNA Polimerase I
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Nucleotídeos
Idioma:
En
Ano de publicação:
2007
Tipo de documento:
Article