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 β (Pol β) functions in base excision repair 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. To probe the mechanism that may underlie E288K's loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol β and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol β when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of 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 because of 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.

Figure 1. The human Pol β variant E288K and the AEDANS-Dabcyl FRET system (Protein Data Bank codes 4KLE and 3ISB)

(A) The four domains of Pol β (4KLE): fingers domain (blue), palm domain (green), the thumb domain (purple), and the 8kDa domain (red). Residue 288 (brown), located at the tip of the fingers domain at the end of helix N, has been mutated to lysine to show the E288K variant. (B and C) Pol β used in this FRET system includes an AEDANS label on residue V303C (pink) and a Dabcyl quencher (light blue) on the DNA substrate. (B) Pol β in the open conformation (3ISB), in which the AEDANS and Dabcyl have a calculated distance of 43.2 Å, which is greater than the Förster radius of 40 Å. (C) In the closed conformation (4KLE), the AEDANS moves closer (33.9 Å) to the Dabcyl and results in a lower signal. Distances are determined using Swiss-Pdb Viewer, Swiss Institute of Bioinformatics.

Figure 2. WT and E288K have similar secondary structures

The ellipticity of a 3 μM protein solution in 10 mM dibasic sodium phosphate buffer was measured from 195–260 nm for AEDANS-labeled WT and E288K Pol β.

Figure 3. WT and E288K display pre-steady-state burst activity

Representative plots are displayed. The biphasic nature of the pre-steady state activity of WT and E288K on extA DNA are evident in plotted data (dots). Curves show the best fit to Equation 1 for each data set. The two bursts shown here are representative of at least ten, but the parameters of the fits shown are WT: k_obs=14±2 s⁻¹, k_ss=0.9±0.1s⁻¹; E288K: k_obs=15±2 s⁻¹, k_ss=1.0±0.1s⁻¹.

Figure 4. FRET demonstrates non-covalent step in E288K correct incorporation mechanism

Using the stopped flow apparatus, a solution of AEDANS-labeled WT or E288K Pol β and ddA (A) or extA DNA (B) DNA was mixed with the given dNTP in the presence of Mg²⁺, excited at 336 nm, and then fluorescence was measured for 10 seconds. Each trace shown is an average of 4 recordings. Data shown (solid curves) is modeled using KinTek Global Explorer. Fits obtained by KinTek Global Explorer are indicated by the dotted curves. Figure S3 shows the Chi Squared analysis of these FRET experiments with ddA and extA DNA and indicates that WT and E288K are best described by a mechanism that includes a non-covalent step before chemistry.

Figure 5. Reverse closing FRET using a trap experiment

A solution containing AEDANS labeled Pol β-ddA-correct dTTP and 10 mM Mg²⁺ in ternary complex was mixed with a 10-fold excess of an unlabeled Pol β-extA binary complex, excited at 336 nM and fluorescence was recorded for 10 seconds. As the unlabeled secondary complex rapidly trapped and sequestered the free and released dTTP, an increase in fluorescence can be observed. Each trace (red for WT and blue for E288K) is an average of at least 4 recordings. Fits obtained by GraphPad Prism are indicated by the black dotted curves. The reverse rates measured using KinTek Global Explorer for WT were k₋₄=0.25s⁻¹, k₋₃=65 s⁻¹, k₋₂=26s⁻¹ and for E288K were k₋₄=0.21s⁻¹, k₋₃=13.3 s⁻¹, k₋₂=64s⁻¹, where k₋₄ corresponds to the reverse non-covalent step, k₋₃ is the reverse of fingers closing and k₋₂ is dNTP dissociation.

Figure 6. FRET with incorrect dNTP and non-extendable DNA shows quenching for E288K but not WT

A solution of AEDANS-labeled WT (A) or E288K (B) Pol β and ddA DNA was mixed with the indicated incorrect dNTP in the presence of Mg²⁺, excited at 336 nm, and fluorescence was measured for 10 seconds. Each trace shown is an average of 4 recordings.

Figure 7. FRET with incorrect dNTP and extendable DNA shows no quenching

A solution of AEDANS-labeled WT (A) or E288K (B) Pol β and extA DNA was mixed with the indicated incorrect dNTP in the presence of Mg²⁺, excited at 336 nm, and fluorescence was measured for 10 seconds. Each trace shown is an average of 4 recordings.

Figure 8. The position of E288 in the ternary state for Pol β

Left shows the position of E288 in the ternary complex (PDB ID 2FMS) in relation to the DNA template strand upstream from the nucleotide insertion site. Right shows the potential position of K288 simulated via mutagenesis in Coot and displaying three standard lysine rotamers from the geometry library. Two of the rotamers would be in position to hydrogen bond to the non-bridging oxygens of the +5 position while contact to the +4 position would likely require a conformational shift or be solvent mediated. Both figures were made using PyMOL.

Scheme 1. Pol β’s mechanism of nucleotide incorporation

Step 1: Pol β first binds single-nucleotide gapped DNA (DNA_n) to form the binary complex. Step 2: The binary complex binds dNTP. Step 3: The fingers domain moves to a closed conformation (β′) Step 4: A non-covalent step occurs. Step 5: The nucleotidyl transfer reaction is carried out to add the dNTP to the single-nucleotide gapped DNA, forming nicked DNA (DNA_n+1) and pyrophosphate (PPi). Step 6: Pol β opens with PPi still bound. Steps 7 – 8: PPi is released followed by DNA product release (Scheme adapted from Towle-Weicksel et. al.).

Scheme 2. Rates obtained from modeling the FRET data with fixed reverse rates using KinTek Global Explorer

Using non-extendable ddA, WT rates are in $\text{red}$ and E288K rates are in $\text{blue}$.