The overall goal of the following experiment is to determine the rates of eight OXO Guine excision and product release for human eight OXO Guine, DNA glycosylate using rapid kinetic techniques. This is achieved by first measuring a steady state time course with low enzyme concentration to determine whether a rapid phase occurs during the first turnover. If the first turnover is rapid, the steady state time course does not extrapolate to zero, but instead reflects the active enzyme concentration as a second step.
The rate of product formation during pre steady state can be measured by rapid mixing and quenching techniques with higher enzyme concentration to observe a significant amount of product during the first turnover. Next, the chemical step can also be monitored under single turnover conditions where substrate is saturated with enzyme. In order to isolate the events at the active site of the enzyme without catalytic cycling, the results show that the product release from the enzyme is rate limiting during steady state based on the observation that the time course does not extrapolate to zero product at zero time.
This method can help answer key questions in the DNA lip repair field. This includes the influence of DNA structure, role of active site residues, effect of metal ions, and cell accessory protein on chemistry and product release. First, prepare the double stranded DNA substrate containing eight OXO G by mixing five prime six fam labeled oligonucleotide with its complimentary strand at a one to 1.2 molar ratio in a kneeling buffer in a 1.5 milliliter micro fuge tube.
Place the tube in a float in boiling water for five minutes. Allow the water to cool slowly to room temperature before removing the tube from the float. Prepare DNA substrate and OG G one solutions separately in reaction buffer in 1.5 milliliter micro fuge tubes on ice.
Note that the concentration of DNA is 400 nano molar and the apparent concentration of Ogg one is 30, 60 90, or 120 nanomolar as determined by a Bradford protein assay After the reaction tubes have been removed from the ice, pre incubate each solution by placing the tube in a heat block set at 37 degrees Celsius for one minute. Next, start the reaction by mixing equal volumes of the DNA substrate and OG one solutions to give final concentrations of 200 nano molar DNA and 15 30 45 or 60 nanomolar OG G one. Remove 10 microliter aliquots at the listed time intervals and quench the reaction by mixing with one microliter of one molar sodium hydroxide as the control for the time courses.
Mix 10 microliters of the reaction mixture without enzyme with one microliter of one molar sodium hydroxide. Place the reaction samples in a 90 degree Celsius heat block for five minutes to cleave the resulting product AP site. After heating, add one microliter of one molar hydrochloric acid to neutralize each sample.
Following this, add 12 microliters of gel loading buffer to each reaction sample, and then place the mixture in a heat block set at 95 degrees Celsius. After two minutes, remove the tube from the heat block and place it on ice. Load five microliters of each sample onto a 15%denaturing poly acrylamide gel.
Once the gel has been run, scan it using an imager that can detect the fluorescently labeled DNA and visualize the substrate and product bands. After imaging the gel, quantify the bands if background cleavage is observed after treatment of the substrate with sodium hydroxide, subtract this background from the measured amount of each reaction product. Next, plot the amount of product formed at each reaction time.
Analyze the raw data using the following equation to determine a knot, the amplitude of the burst and VSS the slope of the linear steady state phase Plot the Y intercept relative to the total protein concentration. Providing a measure of the active fraction of enzyme. Use a linear fit with a zero intercept to provide the correction factor for determining the fraction of active enzyme.
Then plot the steady state relative to the active enzyme concentration, providing the product association rate constant in two separate one five milliliter micro fuge tubes. Prepare 400 nanomolar DNA substrate and 80 nanomolar active ogg one solutions in reaction buffer as described previously. Connect a circulating water bath to the rapid quench flow instrument and set the temperature to 37 degrees Celsius.
Following this, adjust the instrument parameters and select the appropriate reaction loop for the desired time points. According to the manufacturer's instructions, prepare the reaction buffer and 142 sodium hydroxide quench solutions. In 10 milliliter lure lock disposable syringes.
Attach a syringe with sodium hydroxide to the drive ports and set the valves to the load position. Load the drive reservoirs with sodium hydroxide in syringe Q to remove the air bubbles from the syringe. Work the solution back and forth several times.
Next, attach syringes with the reaction buffer to the drive ports and load the buffer in syringes. B, in the same way with all valves in the fire position, lower the stepper motor until it contacts the top of the syringes. With the sample load valves in the flush position, flush the sample loops the reaction loop and the exit line with water and methanol.
Following this, dry the loops and the exit line completely by flushing them with air. Make a hole in the top of a capped 1.5 milliliter tube. Using a 16 gauge needle, attach the tube to the exit line via tubing to collect the quenched reaction.
Next, set the desired reaction. Time in seconds using the keypad. After the stepper motor backs up, position the plungers for syringe B against the stepper motor platform by adding buffer with the disposable syringes attached to the drive ports.
Fill one milliliter lure lock disposable syringes with the DNA substrate and ogg one solutions respectively. With the sample valves in the load position, attach the syringes with DNA substrate and ogg one solutions to each of the sample load ports and then fill the sample loops with the solutions set. All syringe load valves and sample load valves to the fire position start the reaction by pressing the G key.
Once G key is pressed, the stepper motor pushes the top of syringes to start and quench the reaction. After 36 microliters of the reaction mixture has been automatically quenched with 86 microliters of 142 millimolar. Sodium hydroxide collect the sample from the exit line.
Once the reaction is complete, set the syringe load valves to the load position and the sample load valves to the flush position. Flush the sample loops the reaction loop and the exit line with water and methanol and dry the flushed areas completely. Repeat the previous steps for each time point.
To determine a background correction. Perform a control experiment without enzyme using the rapid quench instrument. Fill the DNA sample loop with DNA substrate, but keep the ogg one sample loop empty.
Set the reaction time. Perform the mix and quench using the same conditions as before. Wash the sample loops with two molar sodium hydroxide, two molar hydrochloric acid water and methanol, and then dry the washed lines.
After setting the stepper motor to the home position, switch the syringe load valves to the load position and wash the drive reservoirs with water. After heating the quenched reaction samples, separate the substrate and product DNA by 15%denaturing poly acrylamide gel electrophoresis. Then visualize and quantify the bands on the gel as described.
Previously fit the time courses of product formation by non-linear regression analysis to the following equation with a rising exponential and linear terms providing the first order rate constant K observed the amplitude of the burst, a knot and a linear rate VSS. For the single turnover time course experiment. Prepare 100 nanomolar DNA substrate and 500 nano molar active ogg one solutions in separate 1.5 milliliter tubes.
Using the previously described conditions, prepare the rapid quench flow instrument and conduct the reaction in the same manner and the pre steady state time course experiment. Once the quenched samples have been heated and separated by poly acrylamide gel electrophoresis, visualize and quantify the bands on the gel as described previously. Finally, fit the time courses of product formation to a single exponential to determine the first order rate constant as given in the following equation.
Steady state kinetic analysis was performed by using 200 nanomolar of a DNA substrate and four different concentrations of ogg. One as determined by a Bradford protein assay. Cleaved product and cleaved substrate were separated by poly acrylamide gel electrophoresis.
The time courses of product formation were fit to a linear equation to determine the y intercept, which were 2.2 1115 and 26 nanomolar respectively relative to each protein concentration, the Y intercepts were further plotted relative to each actual protein concentration. The fraction of active enzyme was determined to be 38%from the slope of the line to determine the steady state rate. VSS values determined previously were plotted relative to the Y intercepts.
The slope of the line was 0.0028 per second, which is equivalent to the dissociation rate constant for the product formed by DNA Glycosylate liaise activities. A pre steady state time course was followed using 200 nano molar, DNA substrate and 40 nano molar active OG one. The time courses of product formation can be fit to an equation with rising exponential and linear terms.
Kay observed and koff were determined to be 0.75 and 0.0055 per second respectively. Under single turnover conditions, the eight Oxo g excision reaction was finished within six seconds. The time course of product formation could be fit to a single exponential equation and yielded a K observed of 0.74 per second.
After watching this video, you should have a good understanding of how to isolate the key steps during catalytic cycling and measure the rates of chemistry and enzyme turnover.
