A pre-equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1.
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.
Rothwell
Paul J
PJ
Institute of Structural Molecular Biology, Birkbeck College and University College London, Malet Street, London, WC1E 7HX, United Kingdom.
Waksman
Gabriel
G
Institute of Structural Molecular Biology, Birkbeck College and University College London, Malet Street, London, WC1E 7HX, United Kingdom. Electronic address: g.waksman@ucl.ac.uk.
D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology
D009711:Nucleotides; D004247:DNA; D004256:DNA Polymerase I