期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 282, 期 39, 页码 28884-28892出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M704824200
关键词
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资金
- Wellcome Trust [067879] Funding Source: Medline
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.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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