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 ....

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 rep....

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