Abstract
Author SummaryFaithful replication of genomic DNA by DNA polymerases is crucial for maintaining the genetic integrity of an organism. If DNA becomes damaged, specialized lesion-bypass DNA polymerases are recruited to correct errors in the DNA. A variety of kinetic and structural studies have established a minimal kinetic mechanism common to all DNA polymerases. This mechanism includes several steps involving discrete protein conformational changes. However, the inter-relationship between conformational dynamics and enzymatic function has remained unclear, and identification of the rate-limiting step during nucleotide incorporation has been controversial. In this study, we monitored the directions and rates of motion of domains of a lesion-bypass polymerase during correct nucleotide incorporation. Our study provides several significant findings. First, the binding of a correct nucleotide induces a fast and surprising DNA translocation event. Second, all four domains of the polymerase rapidly move in a synchronized manner before and after the polymerization reaction. Third, repositioning of active site residues is the rate-limiting step during correct nucleotide incorporation. Thus, the motions of the polymerase and the polymerase-bound DNA substrate are tightly coupled to catalysis.
Highlights
Elucidating the mechanism of enzyme catalysis encompasses the identification and characterization of each chemical and conformational intermediate occurring along the reaction pathway [1]
Nucleotide binding induces a significant structural change involving an open-to-close transition of the finger domain for the A, B, and some X-family DNA polymerases [2,3,4,5,6] while ternary complex formation for the Y- and some X-family members [7,8] leads to the subtle repositioning of select active site residues
Design of Two fluorescence resonance energy transfer (FRET) Systems Recently, our crystallographic study of Sulfolobus solfataricus DNA polymerase IV (Dpo4) reports that, upon nucleotide binding, no large-scale domain movements are observed, but local conformational changes occur for active site residues (Y10, Y48, R51, and K159) near the nucleotide binding pocket [8]. To examine if these crystallographic observations are true in solution, we investigated the conformational changes of Dpo4 during a single, correct nucleotide incorporation by monitoring the real-time FRET changes with a stopped-flow apparatus
Summary
Elucidating the mechanism of enzyme catalysis encompasses the identification and characterization of each chemical and conformational intermediate occurring along the reaction pathway [1]. Numerous stopped-flow studies monitoring a single fluorophore, either on DNA (e.g., 2aminopurine) [9,10,11,12,13] or on the finger domain (tryptophan or fluorescent dye) of a DNA polymerase [14,15], have generated interesting but contradictory evidence for this assignment because the fluorescence intensity of a fluorophore can be affected by many factors, thereby complicating data interpretation This assignment of the rate-limiting step has been forcefully questioned due to fluorescence resonance energy transfer (FRET)-based evidence for two A-family DNA polymerases [16,17,18], which shows that the closure rate of the finger domain is too fast to limit correct nucleotide incorporation. It has been hypothesized by us [19,20] and others [16,17,18,21] that the rate-limiting step corresponds to the subtle repositioning of active site residues, which are critical for properly aligning two magnesium ions, the 39-hydroxyl of the primer terminus, the a-phosphate of the incoming dNTP, and the conserved carboxylate residues in the active site
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