Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

Summary

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

Abstract

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

Introduction

The proofreading functions of DNA polymerases during DNA replication are essential to ensure the high fidelity of genetic information that needs to be transferred to progeny^{1,2,3,4,5,6,7}. Being able to assess the contributions of polymerase proofreading exonucleases would clarify the mechanisms safeguarding genetic stability.

Radioisotope labeling and gel-based assays in combination with densitometric analyses of the autoradiograms or phosphor imaging^8,9,10 have traditionally been used to detect proofreading activity of DNA polymerases. While functional, these assays are laborious, expensive, and not amenable to high-throughput formats. In addition, radioisotopes suffer safety issues including waste disposal. Alternatively, proofreading activities have been analyzed by fluorometric techniques. For example, 2-aminopurine (2-AP) can be incorporated into extension products during in vitro polymerase proofreading assays to produce a fluorescent signal^11,12. Unfortunately, these approaches suffer from a low specificity, since 2-AP can pair with both thymine and cytosine. More recent approaches include a sensitive G-quadruplex-based luminescent switch-on probe for a polymerase 3'-5' exonuclease assay¹³ as well as a singly-labeled fluorescent probe for a polymerase proofreading assay that overcomes some of the aforementioned drawbacks¹⁴. Enthusiasm for these fluorometric methods is diminished due to the need for the specific labeling of DNA substrates.

In contrast, a MALDI-TOF MS for DNA analysis has been employed in the PinPoint assay, where the primer extension reactions with unlabeled 4 ddNTPs can be used to identify polymorphisms at a given locus^15,16,17 and has been widely adopted in clinical applications for mutation detections and cancer diagnoses¹⁸. Using these basic principles, we have created a label-free assay for the in vitro determination of DNA polymerase proofreading activity exploiting the high resolution, high specificity, and high-throughput potential of MALDI-TOF MS. Using the E. coli DNA polymerase I Klenow fragment as a model enzyme, dideoxyribonucleotide triphosphates (ddNTPs) as substrates can take a "snapshot" of proofreading products after a single nucleotide extension via MALDI-TOF MS (Figure 1).

Likewise, this method was also developed for a DNA repair assay where primers containing 3' penultimate dI lesions are subjected to a pol I repair assay which mimics endo V nicked repair intermediates. While not fully understood, the endo V repair pathway is the only repair system known to employ the pol I proofreading exonuclease activity for lesion excision^19,20. Using MALDI-TOF MS, we show a clearly defined repair patch where dI can be excised by pol I when occurring in the last 2 nt of the primer before adding the correct complemented nucleotide.

For the study of proofreading and DNA repair, this method is faster and less laborious than previous methods and provides additional information towards mechanism and function.

Protocol

1. Primer/template Preparation

1. Design primers/templates with a balanced G+C content between 40% and 60% as in a sequencing or PCR primer design. Use primers of 18 to 21 nt for an appropriate annealing and better MS signals.
2. Design the template by setting 50 °C as the minimum melting temperature for the duplex region with at least 7 nt of 5'-overhang to separate the signals between the primer and the template.
   NOTE: For example, for the substrate P21/T28 in Table 1, the 21-nt primer is paired with a 28-nt template. Option: the use of alternative nucleic acids such as nuclease-resistant phosphorothioate bonds to replace the last 4 phosphodiester bonds at the 3' end of the template can prevent possible interference by a non-specific 3' end degradation of the templates (e.g., T28S4 in Table 1), although this will add some cost for the template preparation.
3. Using standard desalted oligonucleotides without any further purification is satisfactory for this study. Use oligonucleotide primers/templates at a concentration of 100 pmol/µL in H₂O as a stock and store it at -20 °C. Check the quality and purity of the primers and templates by running the oligonucleotides on MALDI-TOF MS (steps 4 - 7) to ensure unique peak signals and a good signal to noise ratio.
4. Determine the relative MALDI-TOF MS signal intensities (step 7) of the equal molar primers of the relevant sequences in the reaction for calibration and concentration normalization (Figure 2).

2. Proofreading Reactions

Note: The same proofreading reaction condition and protocol applies to a DNA repair of A-I, G-I, C-I, and T-I.

1. Using a T-G mismatch of substrate P21/T28 for the proofreading in Table 1 as an example, dilute primer P21 and template T28 to 12.5 pmol/µL with H₂O; then transfer 12 µL of the diluted primer and 12 µL of the diluted template to a 1.5-mL sterilized microcentrifuge tube.
   NOTE: The amount of template/primer mix is sufficient for 2 reactions; proportionately increase the volume of the reagents accordingly for multiple reactions.
2. Close the tube tightly. Incubate it for 30 min in a covered 65-°C water bath, followed by 30 min in a 37-°C water bath, and finally on ice to ensure a proper annealing.
3. To a 1.5-mL sterilized microcentrifuge tube, add 8 µL of primer and template mix (50 pmol), 2 µL of 10x proofreading reaction buffer, 4 µL of 4 ddNTPs mix (2 mM of each 4 dNTPs), and H₂O up to 18 µL. Flick the tube to mix.
   NOTE: 1x proofreading reaction buffer contains 50 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol, and 10 mM Tris-HCl (pH 7.9 at 25 °C). See the Table of Materials for the commercial source of 10x proofreading reaction buffer. The final concentration of each ddNTP in the reaction is 0.1 mM.
4. Dilute DNA polymerase in ice-cold 1x proofreading reaction buffer to the desired concentration [e.g., to a 1.5-mL microcentrifuge tube, add 4 µL of 1x proofreading reaction buffer and 1 µL of 5-U Klenow polymerase from a commercial source (Table of Materials)]. Store the diluted enzyme on ice at all times.
   NOTE: The amount of diluted DNA polymerase is sufficient for 2 reactions; proportionately increase the volume of the enzymes accordingly for multiple reactions. A volume of 2.0 µL, containing 2.0 units of Klenow polymerase, can proofread more than half of 50 pmol of T-G terminal mismatched P21/T28 in 5 min.
5. Prewarm the microcentrifuge tube with the substrate mix from step 2.3 to the desired reaction temperature (e.g., typically 37 °C for Klenow polymerase and 25 °C for T4 DNA polymerase). Then transfer 2.0 µL of the diluted DNA polymerase from step 2.4 to the prewarmed substrate mixture and flick the tube to mix its contents.
6. Centrifuge the tubes with enzyme and substrate for a few seconds at 3,200 x g at ambient temperature to spin down the components of the reactions. Immediately transfer the reaction to a heating block or a water bath at the desired incubation temperature as in step 2.5.
7. Terminate the reaction
   1. Add an equal volume of buffered phenol, mix it by vortexing for a few seconds, and centrifuge it in a microcentrifuge at 3,200 x g at ambient temperature for 5 min.
      1. Transfer the aqueous phase to a clean microcentrifuge tube and add an equal volume of chloroform, mix it by vortexing for a few seconds and centrifuge it in a microfuge at 3,200 x g at ambient temperature for 3 min. Transfer the aqueous phase to a clean microfuge tube and incubate it at 95 °C for 5 min; then place it on ice.
         CAUTION: Phenol and chloroform are hazardous chemicals. Handling and waste disposal should follow institutional regulations.
   2. Alternatively, use an acid quenching protocol. For this, add 2 µL of 1 M HCl to stop the reaction on ice for 6 min, then add 0.64 µL of 1 M diethanolamine (DEA) to neutralize the pH of the reaction mixture and incubate it at 95 °C for 5 min; then place it on ice.

3. Resin Addition to Eliminate Salt Contamination

1. Using the resin scoop from the commercial desalting kit (see the Table of Materials), take sufficient resin to cover the dimples of the 384-dimple, 6-mg resin dimple plate.
2. Distribute the resin across all the dimples evenly (6 mg/dimple) by scraping across the top of the plate. Recover any excess resin and replace it in the bottle.
3. Transfer the final reaction products from step 2.7 from the microfuge tubes to a 384-well plate. Dispense 16 µL of H₂O to dilute the final reaction products in each well and then seal the plate and centrifuge it to remove any bubbles.
4. Remove the seal, turn the 384-well plate with the reaction products upside down to cover the resin-filled dimple plate.
5. Turn over the well-to-dimple plates sandwich and carefully let the resin drop into the 384-well plate (be sure the 384-well plate is flush against the metal peg on the resin-filled dimple plate).
6. Remove the dimple plate on top, seal the 384-well plate containing the mixtures of the reaction products and resin, briefly centrifuge it at 3,200 x g to remove any bubbles and place it on a rotator for at least 15 min at room temperature for desalting its contents.
7. After the resin cleanup, centrifuge the 384-well plate at 3,200 x g for 5 min to compact the resin to the bottom of the wells.

4. Transfer Reaction Products to a Matrix Chip

1. Set the sample plates in the nanoliter dispenser (nanodispenser, see the Table of Materials).
2. Put the matrix chip (see the Table of Materials) and the 384-well plate from section 3.7 in the corresponding tray.
3. Operate the nanodispenser to spot the reaction products to the chip; push the RUN button on the touch screen of the nanodispenser to start.
4. Check the automated captured image of the sample spots containing saturation information on the screen to ensure the overall spotted volume on the chip is around 5 - 10 nL. Repeat the sample spotting if the volume is insufficient.

5. Setup the Assay Information on the Mass Spectrometry

1. Prepare a file in .xls format containing the anticipated signal information for importing. For example, use the setting of "P21_T28.xls" (Table 2) for the proofreading reaction of P21/T28 in steps 2, 3, and 4.
2. Create and define a new assay in the application program (see the Table of Materials) by right-clicking Import Assay Group in Designer Format and select the .xls file (e.g., "P21_T28.xls" from step 5.1).
3. Establish the target assay plate via the Customer:Project:Plate option tree on the left side of the screen by right-clicking the top of the tree and giving it a file name (e.g., "CTT20171201" represents the assay code and date).
4. Select the 384-well plate type and press OK; a blank plate will appear.
5. Select the appropriate assay (e.g., P21_T28) on the left side of the screen.
6. Assign the selected assay (e.g., P21_T28) for each sample spotted sample position on the chip by highlighting the well and right-clicking to select Add Plex.
7. Prepare a working list of all the tests on the chip in .xlsx format with no header (e.g., 1201.xlsx of Table 3). Import the working list via the Add New Sample Project option.
8. Assign the test in the working list (e.g., from 1201.xlsx) to each position of the P21_T28 assay and choose by right-clicking.

6. MALDI-TOF MS Operation

1. Link the mass spectrometer to the chip using the application program (see the Table of Materials).
2. Select the default setting at the right side of the screen.
3. Fill in the assay name from step 5.3 (e.g., CTT20171201), chip ID at the corner of the chip, and save the settings.
4. Start the MS control program (see the Table of Materials).
5. Press the In/Out button and take out the scout plate. Place the spotted chip from step 4.4 onto the scout plate and press the In/Out button for the spotted chip to enter the mass spectrometer.
6. Click the Acquire button of the application program (see the Table of Materials) to start the mass spectrometry and acquire data.

7. Data Analysis

1. Open the program for the data analysis (see Table of Materials).
2. Browse the tree of the database at the upper left corner and select the chip ID from step 6.3. Open the data file on the right side of the screen.
3. Click the spectrum icon to display the mass spectrum. To crop a specific range of m/z, right-click to select Customization Dialog and in the new window click on X-Axis and set the desired upper and lower limit and press OK to show the specified range of the spectrum.
4. Export the spectrum for record keeping by right-clicking Export.
   NOTE: Using a scale of 1,600 width/1,200 unit is a reasonable size for data inspection on a computer screen.
   1. Select the JPEG file type, click on Destination, select Browse Disc (e.g., flash disc E:), type the file name (e.g., 1201-1.jpg), and then click on Export.
5. For data quantitation, use the cursor and click on the peak to show the peak height at the upper left-hand corner.
   1. Alternatively, print out an exported JPEG file and measure the peak height with a ruler manually.

Results

Templates and primers:

Using the procedure presented here, equal molar synthetic oligonucleotide templates and primers of relevant sequences obtained from commercial sources were checked for their purity and quality (Figure 3A; note the signals matched the designated mass and the low background) as well as for the relationship between the peak intensity and the analyte mass (

Discussion

This study described a step-by-step proofreading activity assay analyzed by the chosen commercial instrument (see the Table of Materials) using MALDI-TOF MS. The major advantages include that the primer and template are label free and easy to perform, allowing for greater flexibility in designing experiments. A stream-lime complete processing of 30 proofreading tests would take 4 h, including 3 h for manually performing the proofreading reactions and their cleanup, while the MALDI-TOF MS analyses using t...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Oligonucleotides	Mission Biotech (Taiwan)
phosphorothioate modified oligonucleotides	Integrated DNA Technologies (Taiwan)
DNA polymerase I, Large (Klenow) Fragment	New England Biolabs, MA	M0210L
Klenow fragment (3'→5' exo-)	New England Biolabs, MA	M0212L
NEBuffer 2.1 (10X)	New England Biolabs, MA	B7202S
2', 3' ddATP	Trilink Biotechnologies, CA	N4001
2', 3' ddGTP	Trilink Biotechnologies, CA	N4002
2', 3' ddTTP	Trilink Biotechnologies, CA	N4004
2', 3' ddCTP	Trilink Biotechnologies, CA	N4005
dATP, dGTP, dCTP, dTTP set	Clubio, Taiwan	CB-R0315
SpectroCHIP array	Agena Bioscience, CA	#01509
MassARRAY	Agena Bioscience, CA
Typer 4.0 software	Agena Bioscience, CA	#10145
Clean Resin Tool Kit	Agena Bioscience, CA	#08040