Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

This is a presentation of two stopped flow methods for measuring the reaction kinetics of mush two mush six A DNA mismatch repair protein mush. Two mush six recognizes base pair mismatches in DNA and signals repair in a reaction fueled by a TP.The first method measures mush two mush six binding to DNA by the increase in fluorescence of a two amino purine base positioned adjacent to a GT MIS pair in DNA. The reactants are mixed in a stopped flow instrument and monitored over time to determine the rate Constance.

The second method measures mush two mush six a TPAs activity using a fluorescent phosphate binding protein reporter. The reactants are mixed in a stopped flow and monitored over time. To determine the rate constants for a TP hydrolysis and phosphate release, the resulting kinetic data elucidate the mechanism of action of mush two mush six in DNA mismatch repair.

Hi, I'm Noah Biro from the Laboratory of MONI in the molecular biology and biochemistry department at Wesleyan University. Hey, I'm DJ J, also from Man's Laboratory at Wesleyan University. And I'm Chris Dset, also from JI'S Laboratory.

Today we'll show you two fluorescent based assays to monitor the kinetics of a reaction in the millisecond timescale. We use this methods in our laboratory to study how DA repair protein match two, meh six binds mismatched the base pairs in DNA and initiates the repair. So let's get started.

Before this procedure can begin, the required protein and DNA reagents must be readily available. Obtain s visier mush two mush six protein that has been previously over expressed in coli and purified by ion exchange and affinity chromatography. To generate the DNA reagents purchase single stranded DNA, modified with the fluorescent nucleotide analog to amino purine, purify the strands by denaturing poly acrylamide gel electrophoresis and ele them to generate mismatched duplex DNA with two amino purine adjacent to a GT mismatch.

Similarly, prepare unlabeled DNA from unmodified single stranded DNA. The DNA sequences used here can be found in the written portion of this protocol. Throughout this procedure, ensure high experimental integrity by using high quality chemicals to minimize fluorescent impurities.

Also filter all stock solutions through a 0.2 micrometer membrane to remove particulates that might clog the stopped flow instrument. To set up for the DNA binding experiments, prepare samples for a final volume of 400 microliters per sample, which is sufficient for about 10 kinetic traces. Keep the samples on ice.

To maintain protein activity, prepare the DNA sample at 0.12 micromolar and the mush two mush six sample at 0.8 micromolar concentration in DNA binding buffer. This will result in final concentrations of 0.06 micromolar DNA and 0.4 micromolar mush. Two M six after one-to-one mixing in the stopped flow.

A stopped flow instrument uses a drive motor to simultaneously and rapidly push the reactant solutions from the drive syringes into a mixing device. The mixed solution then flows into an observation cell for data collection to prepare the instrument. First, ensure that the computer and controller are turned off.

Then turn on the circulating water bath to cool the lamp, ignite the lamp. Set the mono chronometer to the desired excitation wavelength, which is 315 nanometers. For two amino purine, set the SL width to the desired value.

Insert a 350 nanometer cutoff filter at the photo multiplier tube or PMT to collect the two amino purine fluorescent signal. Turn on the controller and computer. Next turn on the circulating water bath.

This fills the water jacket around the drive syringes to maintain the reactance of the desired temperature during the experiment. Finally, execute the stop flow program under the menu for P-M-T-H-V control. Set the PMT voltage, which can be vari initially to adjust PMT sensitivity, but must be kept constant.

During the experiment, measure the dark current to correct for background electrical noise. Next, wash the stopped flow drive syringes and observation cell. Set the sample loading valve to the load position.

Fill a one milliliter syringe with deionized filtered water. Attach the syringe to the loading port located beneath the drive syringe and push water between the two syringes. Repeat the process twice for each.

Drive syringe to be used in the experiment. Then fill the drive syringes with water to wash the observation cell. Switch the sample loading valve to the fire position.

Use the adjust syringe drive command to lower the drive plate and push water through the observation cell and into the exit line. Take care not to push the plunger to the very end of the drive syringe. Finally, raise the drive plate and switch the valve to the load position.

Wash once with reaction buffer and empty the syringes. To begin the DNA binding experiments, transfer each sample from the tube to a fresh one milliliter syringe. Attach each syringe to a loading port and push the solution into the drive syringe.

Remove any air bubbles by manually pushing the solution between the two syringes with intermittent pauses. Then lower the drive plate until it contacts the top of the drive syringes. For data collection, open the set time channels window.

Select the data collection channel mode of data analysis, and the number of traces to be collected. Enter the data collection time during which 1000 data points will be collected. Next, set the valve to the fire position and click the collect data button.

This action initiates mixing of mush two, mush six and DNA, followed by entry of the reaction into the observation. Cell fluorescent emission from two amino purine is monitored over. Collect at least five kinetic traces to obtain a high quality data set and save the file when the experiment is complete.

Open each data file and average multiple kinetic traces. Then save the average data file and export it to a graphing program for analysis. Finally, turn off the lamp, wash the syringes and observation cell extensively with water.

Then turn off the rest of the equipment except for the circulating water bath. This is left on for an additional 15 minutes to cool the lamp. The kinetic data for mush two mush six binding to mismatched DNA are best fit by a single exponential function, which yields a fast rate of association on the order of 10 to the seventh per molar per second analogous stopped flow experiments yield the rate of dissociation of the mush two mush six mismatched DNA complex which occurs very slowly at 0.01 to 0.02 per second.

These results provide a direct measure of the speed and affinity with which mush two mush. Six binds a mismatched base pair during the DNA repair reaction. Next, a stopped flow experiment will be performed to measure the A TPAs activity of mush two mush six.

Under pre-study state conditions for this assay, it is absolutely essential to avoid the use of glass in all stages of reagent preparation, including protein and DNA purification in order to minimize background phosphate levels, phosphate binding protein or PBP that has been purified from e coli and labeled with the MDCC fluorophore is used as a reporter to measure phosphate release following a TP hydrolysis. M-D-C-C-P-B-P is a robust reporter of pre-study state A TP hydrolysis and phosphate release kinetics. It binds pre phosphate rapidly and with high affinity, which results in a large increase in MDCC fluorescence.

Prepare a sample with four micromolar mush, two mush six protein with or without six micromolar mismatched DNA. Prepare another sample with one millimolar freshly dissolved HEP and 20 micromolar M-D-C-C-P-V-P to mop up any contaminating phosphate. Add purine, nucleoside, phosphorylase and seven methyl guine into the samples incubate on ice for at least 20 minutes.

Prepare the stopped flow instrument as described earlier. In addition, mop up any phosphate contaminants from the drive syringes with purine, nucleoside, phosphoryl and seven methyl guine Incubate for 20 minutes. Set the excitation wavelength to 425 nanometers and use a 450 nanometer cutoff filter with the PMT for the detection of M-D-C-C-P-B-P fluorescence.

Load the drive syringes with the samples as described earlier. Set data collection parameters and click on collect data to mix mush two mush six with a TP and M-D-C-C-P-B-P monitor phosphate release by mush two mush six and the coupled increase in M-D-C-C-P-P-P fluorescence Over time, collect at least five kinetic traces to obtain a high quality data set and save the file average multiple kinetic traces and export the data file for analysis as described earlier. Prepare a calibration curve to determine the linear relationship between phosphate concentration and M-D-C-C-P-P-P fluorescence first mix M-D-C-C-P-P-P with different phosphate concentrations under the same experimental conditions in the stopped flow.

Then plot the maximum M-D-C-C-P-P-P fluorescence versus each phosphate concentration for the calibration curve and use the slope to determine phosphate concentration in the mush two mush six reactions plot phosphate concentration versus time and fit the data to an exponential and linear function. This function describes a burst of pre-study state A TP hydrolysis and phosphate release, followed by the linear steady state phase of the reaction. The kinetic data show that mush two mush six hydrolyzes A TP, and releases phosphate rapidly at 1.4 per second in the first catalytic turnover.

This is followed by a slow step in the reaction that limits subsequent turnovers to a sevenfold, slower, steady state catalytic constant of 0.2 per second. However, when mush to mush six is bound to mismatch DNA, the burst of a TP hydrolysis and phosphate release is suppressed. These results show directly that on binding a mismatched base pair and DNA mush too mush six switches to an A TP bound state.

This switch is a critical step in the DNA repair reaction. We have just shown you how to use a stop flow instrument to measure the aines of a reaction in real time. Such measurements have advanced our understanding of the MASH two, MASH six mechanism of action and DNA mismatch repair.

When doing these types of experiments, it is important to remember to use high qualitative agents, including very pure DNA and protein and take special care to award phosphate contamination in the ATPs assay. So that's it. Thanks for watching and good luck with your experiments.