Molecular Biology and Biochemistry Department, Wesleyan University
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Biro, F. N., Zhai, J., Doucette, C. W., Hingorani, M. M. Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein. J. Vis. Exp. (37), e1874, doi:10.3791/1874 (2010).
Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.
A. Measurement of Msh2-Msh6 DNA Binding Kinetics
1. Sample preparation for the Msh2-Msh6 DNA binding kinetics experiment
Preparation of reagents for a fluorescence-based kinetic DNA binding experiment on a stopped-flow is similar to that for an equilibrium experiment on a fluorometer. Indeed equilibrium binding analysis should be performed first to estimate the dissociation constant (KD) for the interaction in order to optimize reaction conditions for kinetic analysis. Stopped-flow experiments require larger quantities of biological materials compared with equilibrium or steady-state experiments; therefore, the approach is most feasible when low milligram amounts of protein are available 11,12 and similar amounts of ligands can be prepared or purchased.
2. Instrument preparation for the Msh2-Msh6 DNA-binding kinetics
A stopped-flow instrument is quite simple in principle. It uses a drive motor to rapidly push two solutions in drive syringes into a mixing device, the mixed solution then flows into an observation cell for data collection (Fig. 3). We use the KinTek stopped-flow, which requires low sample volume, allows single or sequential mixing of reactants, detection of a variety of optical signals, and is very easy to use. Stopped-flow instruments are available from several other manufacturers as well.
3. Msh2-Msh6 DNA binding experiment and data analysis
4. Representative results for Msh2-Msh6 DNA binding kinetics
Kinetic data for Msh2-Msh6 interactions with G:T mismatch DNA, can be fit to a single exponential function and yield a fast binding rate constant kON close to 3 x 107 M-1second-1 (Fig. 4A) and a slow dissociation constant kOFF of 0.012 second-1 (Fig. 4B), which reveals that the Msh2-Msh6 binds a G:T mismatch rapidly and forms a very stable complex with a half life close to 60 seconds 13.
B. Measurement of Msh2-Msh6 ATPase Kinetics
1. Sample preparation for the Msh2-Msh6 ATPase kinetics experiment
2. Instrument preparation for the Msh2-Msh6 ATPase kinetics
3. Msh2-Msh6 ATPase experiment and data analysis
4. Representative results for Msh2-Msh6 ATPase kinetics
The kinetic data show that Msh2-Msh6 hydrolyzes ATP and releases phosphate rapidly at 1.4 second-1 in the first catalytic turnover. This phase is followed by a slow step in the reaction that limits subsequent turnovers to a 7-fold slower steady state kcat of 0.2 second-1 (Fig. 8A). However, when Msh2-Msh6 is bound to mismatched DNA, the burst of ATP hydrolysis and phosphate release is suppressed, and Msh2-Msh6 remains in an ATP-bound state for a longer time.
Figure 1. Purification of S. cerevisiae Msh2-Msh6 from E. coli. Msh2 and Msh6 genes were cloned into pET11a vector and over-expressed in E. coli BL21 (DE3) cells. The protein complex was purified by column chromatography over SP-sepharose, heparin, and Q-sepharose resins. SDS-PAGE analysis shown here contains protein fractions from a Q-sepharose column.
Figure 2. Complementary single-stranded DNAs are annealed to form a duplex containing G:T mismatch with an adjacent Adenosine (for ATPase experiments) or 2-Aminopurine fluorescent base analog of Adenosine (for DNA binding experiments).
Figure 3. Flow of reactants in the KinTek stopped-flow during a single mixing experiment.
Figure 4a. Kinetics of Msh2-Msh6 interaction with a G:T mismatch. Mixing of duplex DNA (0.12 μM) containing a G:T adjacent to 2-Aminopurine with Msh2-Msh6 (0.8 μM) leads to increase in fluorescence over time, and yields a bimolecular rate constant kON = 2.4 107 M-1s-1 for the interaction.
Figure 4b. Kinetics of Msh2-Msh6 interaction with a G:T mismatch. Mixing pre-formed Msh2-Msh6 G:T(2-Ap) complex with excess unlabeled G:T DNA (8 μM) that traps any free Msh2-Msh6 leads to decrease in fluorescence over time, and yields a slow dissociation rate constant, kOFF = 0.012 s-1, indicating a stable complex with a long half life of ~ 60 seconds. Final concentrations: 0.4 μM Msh2-Msh6, 0.06 μM labeled DNA, 4 μM unlabeled G:T DNA trap.
Figure 5. MDCC-PBP preparation. SDS-PAGE analysis of E. coli phosphate binding protein (PBP), purified and labeled with the MDCC fluorophore.
Figure 6. MDCC-PBP response to phosphate. Titration of MDCC-PBP with phosphate (Pi) results in increasing MDCC fluorescence.
Figure 7a. Preparation of the phosphate (Pi) standard curve. MDCC-PBP (20 μM) is mixed with varying amounts of Pi (0 - 8 μM) and fluorescence measured over time until equilibrium is reached. Final concentrations: 10 μM MDCC-PBP, 0 - 4 μM Pi.
Figure 7b. Preparation of the phosphate (Pi) standard curve. Maximum MDCC-PBP fluorescence is plotted versus [Pi] to yield a standard curve. The slope of the line (0.383 μM-1 in this case) is used to convert MDCC-PBP fluorescence into Pi concentration.
Figure 8a. Msh2-Msh6 ATPase kinetics. Msh2-Msh6 (4 μM), in the absence G:T DNA, mixed rapidly with ATP (1 mM) and MDCC-PBP (20 μM) exhibits a burst of ATP hydrolysis and Pi release. Data analysis yields the rate (k hydrolysis = 1.4 s-1) and amplitude (2 μM; 1 site per Msh2-Msh6) of the burst phase, which is followed by a linear, steady state phase at a rate of 0.4 μM s-1 (kcat = 0.2 s-1).
Figure 8b. Msh2-Msh6 ATPase kinetics. Addition of G:T DNA to the reaction (6 μM) suppresses the burst of ATP hydrolysis, stabilizing the complex in an ATP-bound state. Final concentrations: 2 μM Msh2-Msh6, 500 μM ATP, 3 μM DNA, 10 μM MDCC-PBP. Burst kinetics are fit by the following equation: [Pi] = A0e-kt + Vt, where [Pi] is phosphate concentration, A0 is the burst amplitude, k is the observed burst rate constant and V is the velocity of the linear phase (kcat = V/[Msh2-Msh6]).
|Reagent||Stock||Working||Vol, μL||Stock||Working||Vol, μL|
|Msh2-Msh6||5 μM||0.8 μM||64||-||-||-|
|2ApG:T||-||-||-||10 μM||0.12 μM||4.8|
Table 1 DNA binding reaction
|Reagent||Stock||Working||Vol, μL||Stock||Working||Vol, μL||Stock||Working||Vol, μL|
|Msh2-Msh6||20 μM||4 μM||80||20 μM||4 μM||80||-||-||-|
|ATP||-||-||-||-||-||-||50 mM||1 mM||8|
|PBP-MDCC||150 μM||20 μM||53.3||150 μM||20 μM||53.3||-||-||-|
Table 2 ATPase reaction
The example of a DNA mismatch binding protein described here illustrates the power and utility of transient kinetic methods for studying the mechanisms of biological molecules. Stopped-flow measurements on the single turnover time scale provided unambiguous evidence for rapid and specific binding of Msh2-Msh6 protein to a mismatched base pair and formation of a long-lived protein X DNA complex in the reaction 9. Moreover, stopped-flow (and quench-flow) analysis of ATPase activity provided direct evidence for Msh2-Msh6 switching to an ATP-bound conformation after binding mismatched DNA 11,16. This switch is hypothesized to signal DNA repair 17,18. Such high-resolution kinetic data, along with complementary high-resolution structural data are essential to advance understanding of macromolecular function.
This work was supported by an NSF CAREER Award (M.M.H), a Barry M. Goldwater scholarship (F.N.B) and an ASBMB Undergraduate Research Award (C.W.D). The clone for over-expression of PBP was kindly provided by Dr. Martin Webb (MRC, UK).
|37 G||DNA Sequence: 5’- ATT TCC TTC AGC AGA TAT G T A CCA TAC TGA TTC ACA T -3’|
|37 T (2-Ap)||DNA Sequence: 5’- ATG TGA ATC AGT ATG GTA TApT ATC TGC TGA AGG AAA T -3’|
|37 T||DNA Sequence: 5’- ATG TGA ATC AGT ATG GTA T A T ATC TGC TGA AGG AAA T -3’|