The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition

Abstract

Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity approximately 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by approximately 3-fold. Kinetic studies showed that the difference in MutS K(d) for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.

Figure 1. Construct Sequence and Experimental Scheme

Shown here is the basic sequence of the 30-bp construct, kept identical throughout this study. The box denotes the position of the mismatch site X-Y, which can be configured to produce any mismatch combination. The top strand was labeled with fluorescein, the donor, while the bottom strand was labeled with TAMRA, the acceptor, at the underlined positions. The schematic representation shows that the distance between the donor and acceptor shortened upon MutS binding, giving rise to FRET signals.

Figure 2. Equilibrium Titration of Heteroduplexes with MutS

(2A) Equilibrium titration scan of 30-1ΔT with increasing MutS concentration; excitation wavelength = 485 nm. The arrow points in the direction of increasing MutS monomer concentration, from 0 to 300 nM. As the MutS concentration rose, the fluorescein emission peak decreased and the TAMRA peak emission increased. (2B) The intensity of fluorescein peak (517 nm) was taken from each trace and normalized to represent the fraction of heteroduplexes bound by MutS. The binding profile of 30-1ΔT by MutS was fitted to a sigmoidal function, shown by the solid line. The fit provided by a 1:1 binding isotherm, shown by the dashed line, did not describe the data as closely as the fit provided by a sigmoidal function.

Figure 3. Kinetic Association and Dissociation Profiles of MutS

(3A) Kinetic association traces of MutS at three different concentrations with 30-1ΔT are shown here. The intensity of the fluorescein peak decreases at different rates depending on the final concentration of MutS. (3B) The apparent kinetic association rates for two different temperatures are plotted against concentration of MutS. The resulting kinetic association profiles were fitted with a simple quadratic function, indicative of positive cooperativity. (3C) Shown here, kinetic dissociation trace of MutS-30-1ΔT complex was fitted to a double-exponential function, yielding two rates. (3D) The apparent kinetic dissociation rates for two different temperatures are plotted against concentration of non-labeled heteroduplex competitors. The kinetic dissociation rates remained constant regardless of the concentration of competitors.

Figure 4. Non-Specific Binding of MutS: Competitions Assays

Titration of a preformed MutS-30-1ΔT complex with non-labeled non-specific competitors at 21°C: 10-bp homoduplex^† (star), 30-bp homoduplex (filled circle), 90-bp homoduplex^‡ (open box) and 30 nucleotide-long single-strand DNA (filled triangle). For ease of comparison, the 10-bp homoduplex^† is plotted as 1/3 of its actual molar concentration and the 90-bp homoduplex^‡ is plotted as 3 times its actual molar concentration. Thus at any given concentration, the molar concentration of DNA base-pairs would be kept equal across different samples (see text for details). The dashed line indicates the concentration of competitors required to cause half of the preformed specific complex to dissociate. For 30-bp homoduplex, ≈ 21.8 µM was required; for 90-bp homoduplex, ≈ 3.6 µM was required; for 30-ssDNA, ≈ 6.2 µM was required. For 10-bp homoduplex, even at the highest concentration tested, at 67.5 µM, only ≈ 5% of the specific complex became dissociated. The calculated KD-NSP was ≈ 23.1 µM for 30-bp homoduplex, ≈ 3.7 µM for 90-bp homoduplex, and ≈ 6.5 µM for 30-ssDNA.

Figure 5. Effect of AMPPNP on Specific Binding of MutS

(5A) Equilibrium titration of 30-GT by increasing MutS in the presence of different amounts of AMPPNP, ranging from 0 to 2 mM. The Hill plot representing binding profiles of MutS for 30-GT in the absence and in the presence of 2 mM AMPPNP is shown in the inset. The Hill coefficient decreased from 2.4 to 1.4. (5B) The apparent kinetic association rates of MutS for 30-1ΔT in the absence of AMPPNP (filled circle) and in the presence of 1 mM AMPPNP (open box). The apparent kinetic association rates of mutS-A842E in the absence of AMPPNP (filled triangle) and in the presence of 1 mM AMPPNP (star). (5C) The apparent kinetic dissociation rates of MutS-30-1ΔT complex in the absence and in the presence of 1 mM AMPPNP. (5D) EMSA: Lane 1: 30-ssDNA, Lane 2: 30-1ΔT, Lane 3: 30-1ΔT complexed with 1µM MutS, Lane 4, 30-1ΔT complexed with 1 µM mutS-A842E, (Lane 5~8: with 1 mM AMPPNP) Lane 5: 30-1ΔT, Lane 6: 30-1ΔT complexed with 1µM MutS, Lane 7, 30-1ΔT complexed with 1 µM mutS-A842E, Lane 8: 30-ssDNA.

Figure 6. Effect of AMPPNP on Stoichiometric Titration of Heteroduplexes by MutS and mutS-A842E

High concentrations of heteroduplex (600 nM ≈ 10-fold KD) was titrated by increasing concentrations of either MutS or mutS-A842E, a single-point mutation deficient in tetramer formation. The titrations were carried out both in the absence and in the presence of 1mM AMPPNP. The initial linear phase is evident for both sets of titration data, as is the plateau region near the end. A correction term was applied to the slope of the initial data points (MutS ≤ 1000 nM) to calculate the value of stoichiometry of MutS-heteroduplex complex (see Materials and Methods). The stoichiometry of MutS-30-1ΔT was determined to be 3.58 in the absence of nucleotide cofactors, and 3.7 in the presence of 1 mM AMPPNP. Whereas the stoichiometry of mutS-A842E-30-1ΔT was determined to be 2.27 in the absence of nucleotide cofactors, and 2.21 in the presence of 1 mM AMPPNP.