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1.
Figure 4

Figure 4. In vitro reconstitution of the long-patch NIR pathway using 5-OH-Hyd•G duplex DNA substrate.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

3′-[α-32P]-ddATP-labelled 5OH-Hyd•G oligonucleotide duplex was incubated with human proteins in the reaction buffer for 1 h at 37°C. Lanes 1–10, reconstitution reactions in the presence of indicated proteins; lanes 11–20, same as 1–10 but treated with Nei. The arrows denote the position of the 23-mer, 14-mer, 13-mer and 12-mer fragments. Note that “23-mer” denotes the position of both the 3′-[32P]-labelled 5OH-Hyd•G substrate and repaired C•G oligonucleotide duplex. For details see Materials and Methods.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
2.
Figure 3

Figure 3. Activity of various E. coli and human DNA glycosylases on pyrimidine-derived hydantoins.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

5 nM of 5′-[32P]-labelled oligonucleotide duplexes were incubated with 50 nM of DNA glycosylase for 30 min at 37°C. For the mono-functional DNA glycosylases a light piperidine treatment was performed to reveal potential AP sites. (A) 5OH-Hyd•G oligonucleotide duplex; (B) 5OH-5Me-Hyd•A oligonucleotide duplex; (C) Graphic representation of the means of enzymatic activities from three independent experiments. The background values representing control oligonucleotides degradation in absence of enzyme (treated or not with piperidine) were subtracted. For details see Materials and Methods.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
3.
Figure 2

Figure 2. MALDI-TOF MS analysis of the mixture of oligonucleotides arising from the incubation of the 22-mer DNA duplexes containing hydantoin residues with AP endonucleases.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

Typically, 40 pmol of the lesion containing oligonucleotide duplexes were incubated with either 3 units of Nfo or 170 ng of APE1 or 100 ng of NEIL1 in the appropriate reaction buffer (10 µL) at 37°C for 30 min. The products were desalted on a MicroSpin G-25 column, prior subjection to the MALDI-TOF MS measurements. (A) Treatment of 5OH-Hyd•G duplex with Nfo. (B) Treatment of 5OH-5Me-Hyd•A duplex with Nfo. (C) Treatment of 5OH-Hyd•G duplex with APE1. (D) Treatment of 5OH-5Me-Hyd•A duplex with APE1. (E) Treatment of αdA•T duplex with APE1. (F) Treatment of 5OH-5Me-Hyd•A duplex with NEIL1. Peaks corresponding to complementary strand are indicated in grey. The sequence of complementary strand corresponding to a double charged species ([M-H]2− = 3383) is shown in truncated form due to space limitation.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
4.
Figure 1

Figure 1. Pyrimidine hydantoins are substrates for the NIR and BER pathways.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

(A) Chemical structures of 5-hydroxyhydantoin-2′-deoxynucleoside (5OH-dHyd) and 5-hydroxy-5-methylhydantoin-2′-deoxynucleoside (5OH-5Me-dHyd). (B) Denaturing PAGE analysis of the cleavage products after incubation of the 3′-[32P]-labelled 5OH-Hyd•G (lanes 1–7) and 5OH-5Me-Hyd•A (lanes 11–18) duplex oligonucleotides with the DNA glycosylases/AP lyases (10 nM) and AP endonucleases (0.5 nM) of different origins. Lanes 1 and 11, control no enzyme; lanes 4 and 14, Nfo; lanes 5 and 14, APE1 under NIR conditions; lanes 6 and 15, APE1 under BER conditions; lanes 7 and 17, Apn1; lanes 2 and 12, Fpg; lanes 3 and 13, Nth; size markers: lane 8, 14-mer fragment with 5′-terminal pdC nucleotide; lanes 9 and 18, 14-mer fragment with 5′-terminal pT nucleotide; lane 10, 13-mer fragment with 5′-phosphate. For details see Materials and Methods. The arrows and “<” symbol denote the position of the 23-mer, 13-mer and 14-mer fragments, respectively.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
5.
Figure 5

Figure 5. DNA repair activities towards pyrimidine-derived hydantoins in E. coli cell-free extracts.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

3′-[α-32P]-ddATP-labelled oligonucleotide duplexes were incubated with either 3 µg of cell-free extract or limited amount of a purified protein in the standard DNA glycosylase reaction “BER+EDTA” buffer for 30 min at 37°C. (A) Denaturing PAGE analysis of the reaction products. Lane 1, control 5OH-Hyd•G with no enzyme; lanes 2–8, 5OH-Hyd•G incubated with extracts; lanes 9–12, 5OH-Hyd•G incubated with the purified proteins; lane 13, control 5OH-5Me-Hyd•A with no enzyme; lanes 14–20, 5OH-5Me-Hyd•A incubated with extracts; lanes 21–24, 5OH-5Me-Hyd•A incubated with the purified proteins. (B, C) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A. DNA glycosylase (BER) and AP endonuclease-catalyzed (NIR) incisions were calculated by measuring amount of 13-mer and 14-mer products, respectively. The background values representing control oligonucleotides degradation in absence of enzyme in lanes 1 and 13 were subtracted. “WT+P” indicates that the expression of Nfo was induced by the exposure of cell culture to 0.25 mg/mL of paraquat. For details see Materials and Methods.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
6.
Figure 6

Figure 6. APE1-catalyzed nucleotide incision activity towards pyrimidine-derived hydantoins in HeLa cells extracts.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

3′-[α-32P]-ddATP-labelled oligonucleotide duplexes were incubated with either 0.5 µg of HeLa cells extract or a purified protein either under “NIR+Zn2+” (lanes 2–3, 6 and 9–10, 13) or under “BER+EDTA” conditions (lanes 4–5, 7 and 11–12, 14) for 1 h at 37°C. (A) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lanes 2 and 4, 5OH-Hyd•G incubated with extracts from HeLa cells treated with the non-specific siRNA (100 nM); lanes 3 and 5, 5OH-Hyd•G incubated with extract from HeLa cells treated with the APE1-specific siRNA (100 nM); lane 6, 5OH-Hyd•G treated with 1 nM APE1; lane 7, 5OH-Hyd•G treated with 5 nM NEIL1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. (B) Western blot analysis of siRNA-induced down-regulation of the APE1 expression in HeLa cells. (C) Graphic representation of the mean values of DNA glycosylase (BER) and AP endonuclease (NIR) activities in extracts, representing amounts of the 13-mer and 14-mer products, respectively. The cleavage activities in each cell free extract were normalized to the relative densitometry values of actin bands on western blot in panel B. For details see Materials and Methods.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.
7.
Figure 7

Figure 7. NTH1-catalyzed DNA glycosylase activity towards pyrimidine-derived hydantoins in HeLa cell extracts.. From: New Insights in the Removal of the Hydantoins, Oxidation Product of Pyrimidines, via the Base Excision and Nucleotide Incision Repair Pathways.

5′-[32P]-labelled oligonucleotide duplexes were incubated with HeLa cell extracts under “BER+EDTA” condition. (A) Denaturing PAGE analysis of the reaction products. Lane 1, control non-treated 5OH-Hyd•G; lane 2, as 1 but incubated with extract from HeLa cells treated with 400 nM of the non-specific siRNA; lane 3, as 1 but incubated with the extract from HeLa cells treated with 100 nM NTH1-specific siRNA; lane 4, as 3 but using 400 nM of NTH1-specific siRNA; lane 5, as 1 but with 10 nM NTH1; lane 6, as 1 but with 5 nM NEIL1; lane 7, as 1 but with 1 nM APE1; lanes 8–14, same as 1–7 but with 5OH-5Me-Hyd•A as a substrate. (B) Western blot analysis of the siRNA-induced down-regulation of NTH1 expression in HeLa cells. Lane 1, control HeLa cells tansfected with 400 nM of the non-specific siRNA; lane 2, HeLa cells tansfected with 100 nM of NTH1-specific siRNA; lane 3, same as 2 but 400 nM siRNA; lane 4, the purified truncated recombinant ΔN-NTH1 protein. (C) Graphic representation of the mean values of DNA repair activities on 5OH-Hyd•G and 5OH-5Me-Hyd•A in extracts. For comparison DNA repair activities on DHU•G substrate were also shown. The cleavage activities in each cell-free extract were normalized to the relative densitometry values of the actin bands on the western blot in panel B. The arrows denote the position of the 9-mer cleavage fragments containing 3′-dRP residue (9-dRP), 3′-hydroxyl group (3′-OH) and 3′-phosphate residue (3′-P), generated by NTH1, APE1 and NEIL1, respectively. For details see Materials and Methods.

Modesto Redrejo-Rodríguez, et al. PLoS One. 2011;6(7):e21039.

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