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

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 (Figure 2A). The peak heights were measured and calculated (Figure 2B) for the MS data normalization.

Reaction condition and mass spectra:

The use of single base primer extension reactions coupled with MALDI-TOF MS for genotyping has previously been shown to be feasible using standard ddNTPs terminators¹⁷. The proofreading assay using standard ddNTPs is similar to a singleplex reaction of genotyping but is much simpler and very easy to perform to obtain clean and reliable results.

The MS spectra covered all the signals generated from both primers and templates (Figure 3). The proper design of the primers and templates generated a well-separated signal profile for both the templates and the primers (Figure 3). According to the m/z value, signals from templates and their non-specific partial degradation products (m/z > 7,000) were well separated from the mismatched primers and proofread products (m/z < 7,000 in Figure 3). If necessary, introducing nuclease resistant phosphorothioate bonds to replace the last 4 phosphodiester bonds at the 3' end of the template (Table 1; T28S4/P21) can reduce non-specific template hydrolysis (Figure 3G; d27).

In the absence of dNTPs, KF has a robust 3'-exonuclease and is capable of degrading both the primer and the template in vitro²¹. Like the native 4 dNTPs, the addition of the 4 ddNTPs prevents a 3' end degradation by KF (Figure 3C and 3E). Likewise, a one nucleotide extension of the 3'-ends adds increased stability to the primer. Using pol I proofreading conditions as previously described²⁰ with 10 U (46 pmol) KF, more than 80% of the 50-pmol mismatched substrates were corrected in 5 min (Figure 3C, 3E, and 3G).

Intermediates and mechanism analysis:

MALDI-TOF MS resolution can be as fine as 1 dalton; indeed, all substrates, products, and intermediates with mass changes in the reaction can be resolved in the same MS spectrum of the selected range. For example, in a terminal T-G mismatch, the mismatched G at the primer terminus was corrected with the incorporation of ddA with an m/z difference of only 32. Such an m/z difference can be easily identified on the MS spectrum in an m/z range from 5,000 to 7,000 (Figure 4A). The change of each individual signal can be calculated by summing up all signals from the mismatched primer, excision intermediates, and proofread products as 100%. For example, in the T-G proofreading reaction, the corrected product relative to the excision product was 23% versus 17% at 5 min, 31% versus 18% at 10 min, and 69% versus 14% at 20 min (Figure 4A).

When a penultimate T-G mismatch is present in the primer, the last 2 nt should be excised followed by a ddA addition to complete the correction. Indeed, the correct products increased with time from 12% at 5 min, 28% at 10 min, to 45% at 20 min of the reaction. In addition, two forms of excision products for a penultimate T-G proofreading, the 1-nt excision intermediate and 2-nt excision products, were easily resolvable (Figure 4B).

Proofreading kinetics with single dNTP:

For E. coli DNA pol I, ddNTPs are not suitable substrates and should be regarded as inhibitors²². Alternatively, using a single 'correct' dNTP in the proofreading reaction instead will also suppress non-specific 3'-5' exonuclease activity and the generation of single nucleotide extension products suitable for MS analysis (Figure 5B). The proofreading rate obtained from reactions containing a single dCTP is about two times higher than reactions containing the 4 ddNTPs (Figure 5A, 5B, and 5C). Thus, the use of a single 'correct' dNTP in the proofreading assay yields a better result without the inhibitory effects of ddNTPs. This approach should provide greater sensitivity for proofreading kinetic analyses.

Proofreading of internal mismatches:

MS proofreading assays using KF for internal mismatches and the identification of proofread products are straightforward (i.e.,the proofreading exonuclease excises nucleotides from the 3' end to the internal mismatch of the primers), followed by the addition of matched ddNMP to the excision products via the polymerase function generating proofread products shorter than the primer substrates (Figure 6). A clear trend of proofreading efficiency was observed, where KF efficiently proofread mismatches at the last, penultimate, 3^rd and 4^th nucleotides from the 3' end of the primers (Figure 6; MM1, 2, 3, and 4). Mismatches at 5 nt from the 3' end of the primer were corrected partially with < 15% of mismatch correction, and > 15% of the primer showed an extension without proofreading (Figure6; MM5). KF failed to proofread mismatches positioned at 6, 7, 8, and 9 nt from the 3' end of the primer (Figure 6; MM6, 7, 8, and 9) but still extended a significant proportion of substrates (Figure 6; PE of MM6, 7, 8, and 9).

Repair of deoxyinosine lesion:

In E. coli, deoxyinosine in DNA is primarily processed by the endo V repair pathway^20,23. Endo V initiates the reaction by an incision at the second phosphodiester bond 3' of the dI lesion. The strand break generated is believed to be used by pol I for a subsequent lesion excision and DNA resynthesis²⁴. This procedure was applied to test pol I repair substrates containing dI at the 3' penultimate site (Table 1; A-I, C-I, G-I, and T-I) to validate this hypothesis. The repair of the 3' penultimate dI is analogous to the proofreading of the penultimate T-G mismatch (Figure 4B and 6; MM2 entry), in which the last 2 nt should be removed followed by an addition of a correct ddNMP to complete the repair. MS patterns clearly showed pol I corrected heteroduplexes of A-I, G-I, and T-I, albeit with different efficiencies (Figure 7; RP signals). However, the C-I substrate was refractory to the pol I repair (Table 1 and Figure 7D and 7E; very low RP) and demonstrated the primer extension to a great extent (Figure 7D; PE signal).

Figure 1. Model system for proofreading assay. A mismatched primer was annealed as indicated to a template forming a terminal mismatch (bold). Proofreading exonuclease of DNA polymerase would detect and remove the mismatched nucleotide forming an excision product. In the presence of a DNA polymerase and four ddNTPs, the excision product is extended by a single base complementary to the previous mismatched site. When subjected to MALDI-TOF, the difference in mass between the mismatched primer, the excision products, and the proofread products can be resolved. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Figure 2. Peak intensity and oligonucleotide mass. Eight oligonucleotides from 17 to 24 nt with sequences complementary to the 3' end of T28 (Table 1) were tested. (A) A mixture of 50 pmol of each oligonucleotide in 40-µL solutions was subjected to an MS analysis. (B) The peak height of 17 nt was used as 100% for the calculation. The relative signal intensity for each oligonucleotide was the average of six determinations and the error bars represent 1 standard deviation. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Figure 3. Effect of various template design. Proofreading reactions were assessed with the P21 primer forming a terminal T-G mismatch with different templates. The proofreading assay was performed with 10 U (43 pmol) Klenow polymerase, 50 pmol substrate, and 4 ddNTPs at 37 °C for 5 min. (A) This panel shows a P21/T32 T-G substrate without enzyme. (B) This panel shows a primer extension of a P21/T32 T-A substrate by 3' exonuclease deficient Klenow. PE = primer extension product. (C) This panel shows a proofreading of the P21/T32 T-G substrate. PP = proofreading product; d26, d27, d28 = 3' end-degraded template. (D) This panel shows a P21/T28 T-G substrate without enzyme. (E) This panel shows a proofreading of the P21/T28 T-G substrate. PP = proofreading product; d26, d27 = 3' end-degraded template. (F) This panel shows a P21/T28S4 T-G substrate without enzyme. (G) This panel shows a proofreading of the P21/T28S4 T-G substrate. PP = proofreading product; EP = excision products; d27 = template degradation by-product. (This figure is adapted from Su et al.²⁵ with permission). Please click here to view a larger version of this figure.

Figure 4. Time course analysis for excision products and proofreading products. Proofreading assays were performed with 2 U (8.6 pmol) KF and 50 pmol substrates as described in the protocol. The reactions were quenched at the indicated times. (A) This panel shows a proofreading of a 3' terminal T-G mismatch; EP = proofreading excision product; d18 & d19 = excess excision by-products; PP = proofreading products; P21 = mismatched primer. (B) This panel shows a proofreading of a 3'-penultimate T-G mismatch; EI-1 = excision intermediate; EP-2 = proofreading excision products; d18 = excess excision by-product; PP = proofreading products; P21 = mismatched primer; Na = sodium ion bound nucleotides. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Figure 5. Proofreading reaction with 4 ddNTPs versus a single dNTP. Proofreading assays were performed with 0.1 U (0.43 pmol) KF and 50 pmol substrates as described in the protocol. The reactions were quenched at the indicated times. (A) This panel shows a proofreading of a 3' terminal G-G mismatch with 4 ddNTPs; EP = proofreading excision product; PP = proofreading resynthesis products; P21G = mismatched primer. (B) This panel shows a proofreading of a 3' terminal G-G mismatch with dCTP; PP = proofreading resynthesis products; P21G = mismatched primer. (C) The correction levels are the average of three determinations and the error bars represent 1 standard deviation. Na⁺ = sodium ion bound nucleotides. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Figure 6. Proofreading of internal mismatches. Proofreading assays were performed with 2 U (8.6 pmol) Klenow polymerase and 50 pmol substrates in 37 °C for 20 min as described in the protocol. Blank = enzyme blank of an MM1 substrate. MM1 = a P21/T28 T-G substrate; MM2 to MM9 = substrates with internal mismatches, the numbers indicate the mismatch position relative to 3' end. P21 = mismatched primers; PR = proofread products; PE = extended primers; d19 and d20 = excess excision by-products, the numbers represent the size of nucleotides. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Figure 7. Repair of 3' penultimate deoxyinosine lesions. Repair assays were performed with 2 U Klenow polymerase and 50 pmol substrates in 37 °C as described in protocol step 2.3. The reactions were quenched at the indicated times. These panels show reactions with (A) T-I substrate; (B) G-I substrate; (C) A-I substrate; and (D) C-I substrate. RP = repair products; EP = excision products; PE = primer extension products, Na⁺ = sodium ion adducts. (E) The correction levels for T-I, G-I, and A-I were the average of three determinations and the error bars represent 1 standard deviation. The correction levels for C-I were the average of two determinations. (This figure is adapted from Su et al.²⁵ with permission.) Please click here to view a larger version of this figure.

Table 1. Proofreading substrates and correction efficiency by Klenow fragment. This table shows the bold 4-nucleotides at the 3'-end of the template containing phosphorothioate modifications. The mismatched base pairs are in bold. The proofreading assays were performed with 2 U (8.6 pmol) Klenow polymerase, 50 pmol substrate, and 4 ddNTPs at 37 °C for 10 min. * These values are the lower limits for the proofreading level because the background noise prevents accurate estimates of < 1% of the total mass signals observed. (This figure is adapted from Su et al.²⁵ with permission.)

Table 2. T28_P21.xlsx file. The setting was used for Figure 3D, 3E, 4A, and 5A.

Table 3. 1201.xlsx file. The setting was used for 7 reactions in Figure 4A.