Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity.

Abstract:

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca (2+) instead of Mg (2+) as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

Polymerases:

Topics:

Methods, Kinetic Parameters, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Joyce CM2008 Methods Featured Fluorescence Spectroscopy
Klenow fragment Joyce CM2008 Cloned or native Cloned in E. coli
Klenow fragment Joyce CM2008 Tagged Yes
Klenow fragment Joyce CM2008 Tag Name N-His6
Klenow fragment Joyce CM2008 Full length or truncated Full length
Klenow fragment Joyce CM2008 kcat 140 /second Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Stopped flow (FRET assay of fingers-closing); Experimental conditions: pH (pH 7.5), Mg++ (5mM), Temp (22°C)

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