Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases.
Various kinetic studies on nucleotide incorporation by DNA polymerases have established that a rate-limiting step occurs that is crucial in the mechanism of discrimination between correct versus incorrect nucleotide. Crystallographic studies have indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. However, there is no direct evidence to support this hypothesis. In order to investigate whether or not the open-to-closed conformational transition affecting the fingers subdomain is rate limiting, we have developed a fluorescence resonance energy transfer (FRET) system, which monitors motions of the fingers subdomain. We establish that the closing of the fingers subdomain is significantly faster than the kinetically determined rate-limiting step. We propose that the rate-limiting step occurs after the closing of the fingers subdomain and is caused by local reorganization events in the active site.
Rothwell
Paul J
PJ
Institute of Structural Molecular Biology, Birkbeck College and University College London, Malet Street, London WC1E 7HX, United Kingdom.
Mitaksov
Vesselin
V
Waksman
Gabriel
G
D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009038:Motion; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D019914:Taq Polymerase / Q000737:chemistry* / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase