Defective Nucleotide Release by DNA Polymerase β Mutator Variant E288K Is the Basis of Its Low Fidelity

Abstract

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase beta (Pol β) functions in Base Excision Repair (BER) to fill in single-nucleotide gaps, and variants of Pol β have been associated with cancer. Specifically, the E288K Pol β variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. In order to probe the mechanism that may underlie E288K’s loss of fidelity, a fluorescence resonance energy transfer (FRET) system was employed which utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate. Our results show that E288K utilizes an overall mechanism similar to wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of fingers closing combined with a slower rate of nucleotide release compared to WT Pol β. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol β. Taken together, our results suggest that E288K Pol β incorporates all dNTPs more readily than WT due to an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure.

DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

“…We observed an increase in the rate of fingers closing with increasing concentrations of the complementary dNTP, as expected for an induced fit mechanism. Previous studies (18,19) found a rapid fingers closing rate of 98 s -1 , close to the maximum rate of 85 s -1 that we determined from our fit. The opening rate is concentration-dependent as well, and decreases with increasing concentrations of dTTPs.…”

“…The distance calculated from structures 1BPX and 1BPY is significantly shorter (6.0 nm). We used the peak shift to determine the dissociation constant Kd, describing the binding affinity of DNA and Pol β, and obtained Kd = 19 ± 2 nM, in good agreement with previously published values (19,29). A trace of a single DNA molecule at [wt Pol β] = 10 nM shows excursions between the low and high FRET states (Fig.…”

“…Our experiments with wt Pol β showed substrate binding with a Kd of 19 ± 2 nM. Other studies have also reported values in the low nanomolar range for gapped DNA constructs: fitting stopped-flow data resulted in a Kd of 5 nM (19), and a titration based on single-turnover analysis at different DNA concentrations revealed a Kd of 22 nM (29). Surprisingly, strong DNA bending upon polymerase binding, as predicted by various structures resolved with X-ray crystallography, does not occur to the expected extent suggesting that the bend angle in solution is larger than 90°.…”

“…The Kd of the incoming nucleotide (1 / K1 = 56 µM) is higher than determined before using stoppedflow and chemical quench analysis (Kd = 2.5 µM) (18,19). The reason for this difference is that we fitted our observations to a model that accounts for fingers opening and closing in presence and absence of dNTPs.…”

“…Non-complementary nucleotides were not found to induce fingers-closing, leading the authors to hypothesize that discrimination between correct and incorrect nucleotides already takes place before fingers-closing. In later work, the authors showed that a low-fidelity Pol β mutant found in cancer cells exhibits altered fingers dynamics (19).…”