|Year : 2016 | Volume
| Issue : 1 | Page : 42-45
2,2,4-Triamino-5(2H)-oxazolone is a weak substrate for nucleotide excision repair
Katsuhito Kino1, Kaoru Sugasawa2, Hiroshi Miyazawa1, Fumio Hanaoka3
1 Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan
2 Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Kobe, Japan
3 Department of Life Science, Faculty of Science, Gakushuin University, Tokyo, Japan
|Date of Web Publication||19-Feb-2016|
Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo - 171-8588
Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki, Kagawa - 769-2193
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: 2,2,4-Triamino-5(2H)-oxazolone (Oz) is a guanine lesion produced by reactive oxygen radicals and photosensitized oxidation. This nucleobase is a potentially mutagenic lesion, and is removed by several base excision repair enzymes. Our purpose is to analyze whether Oz is the substrate of nucleotide excision repair (NER). Materials and Methods: A lymphoblastoid cell line from the patient with xeroderma pigmentosum (XP) complementation group C (XP3BE) was used. Cell-free NER reactions with covalently closed circular DNAs containing the Oz lesion were performed using the XP3BE whole cell extracts with or without the XPC-RAD23B complex. In addition, DNA fragments (180 bp in length) containing the Oz lesion were used for binding reactions with the XPC-RAD23B complex. Results: We analyzed the cell-free NER activity on Oz and the binding affinity of XPC-RAD23B, which initiates NER. Human cell-free NER activity on Oz was detected, though the reactivity to Oz was lower than that on ultra violet (UV)-induced pyrimidine (6-4) pyrimidone photoproduct (6-4PP). Also, binding of XPC-RAD23B with Oz was lower than that with 6-4PP. Conclusion: Because of the low binding affinity of Oz for XPC-RAD23B, NER efficiency on Oz is very low. Therefore, general NER is not the appropriate repair system for Oz.
Keywords: Guanine oxidation, nucleotide excision repair, oxazolone, XPC-RAD23B
|How to cite this article:|
Kino K, Sugasawa K, Miyazawa H, Hanaoka F. 2,2,4-Triamino-5(2H)-oxazolone is a weak substrate for nucleotide excision repair. J Pharm Negative Results 2016;7:42-5
|How to cite this URL:|
Kino K, Sugasawa K, Miyazawa H, Hanaoka F. 2,2,4-Triamino-5(2H)-oxazolone is a weak substrate for nucleotide excision repair. J Pharm Negative Results [serial online] 2016 [cited 2017 Nov 20];7:42-5. Available from: http://www.pnrjournal.com/text.asp?2016/7/1/42/177068
| Introduction|| |
Endogenous and exogenous oxidative stress causes DNA damage; however, there are several enzymes that can repair DNA damage. , Among the four nucleobases, guanine is the most susceptible to oxidative damage. Although 8-oxo-7,8-dihydroguanine (8oxoG) is known to be a guanine oxidation product and a typical oxidation marker, 8oxoG can be further oxidized. 2,5-Diamino-4H-imidazol-4-one (Iz) is produced from guanine and 8oxoG by oxidation. ,, However, Iz is hydrolyzed to 2, 2, 4-triamino-5(2H)-oxazolone (Oz) [Figure 1]a.  There are 2-6 molecules of Oz per 10 7 guanines in liver DNA. 
|Figure 1: Damaged DNA substrates used in this study. (a) Chemical structures of Oz and 6-4PP. (b) The sequences of the DNA substrates. The blunt-ended 180 bp DNA substrates were obtained by digestion with BssHII followed by 3'-end filling with T4 DNA polymerase|
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We recently reported that eukaryotic DNA polymerases alpha, delta, and epsilon almost exclusively insert guanine opposite Oz. , By ab initio calculation, we previously predicted that the Oz:G base pair has two hydrogen bonds, and it is comparatively stable, ,, and that Oz:G is significantly more thermodynamically stable than Oz:C based on determination of T m values.  Extension using DNA polymerases showed that the extension beyond Oz:G is more efficient than that beyond Oz:C.  Thus, Oz will certainly cause G: C-C: G transversions in eukaryotes, and the repair of Oz from the Oz:C base pair is required to prevent point mutations before guanine is incorporated opposite Oz.
Previously, Escherichia More Details coli Nei and Nth enzymes were shown to excise Oz from dsDNA oligomers with similar efficiencies regardless of the type of base in the opposite strand. , Also, the activities of human NEIL1 and NTH1 on Oz were the same as those of Nei and Nth enzymes.  In addition, human OGG1 and AP endonuclease 1 (APE1) were unable to excise the Oz residue, , and another base excision repair enzyme single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) showed no reactivity to the Oz residue (data not shown). Chlorella virus pyrimidine dimer glycosylase and E. coli endonucleases IV and V have moderate activities on Oz compared with each positive control lesion.  Thus, we still have not found enzymes that correctly and efficiently recognize Oz opposite cytosine.
Nucleotide excision repair (NER) is an important DNA repair system that can eliminate a wide variety of lesions, such as ultra violet (UV)-induced cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) [Figure 1]a, as well as intrastrand crosslinks and bulky adducts induced by chemical carcinogens.  Xeroderma pigmentosum (XP) impairs NER activity. The gene responsible for the NER defect of the patients with XP genetic complementation group C encodes XPC protein, which exists in vivo as a heterotrimeric complex with one of the two human homologs of Rad23 in budding yeast (RAD23A or RAD23B) and centrin 2. , Although centrin 2 substantially enhances the DNA damage recognition activity of XPC-RAD23B,  biochemical studies have revealed that the XPC-RAD23B heterodimer is sufficient to bind certain types of DNA lesions with specificity and initiates NER in vitro.  It is unknown whether Oz is the substrate of NER. Here, we analyzed cell-free NER activity on Oz and the binding specificity of XPC-RAD23B to Oz-containing oligonucleotides in vitro.
| Materials And Methods|| |
MgCl 2 , DTT, NaCl, glycerol, NaH 2 PO 4•2H 2 O, Na 2 HPO 4•12H 2 O, and Triton X-100 were purchased from Wako Pure Chemical Industries (Osaka, Japan). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and ethylenediaminetetraacetic acid (EDTA) were purchased from Dojindo Laboratories (Kumamoto, Japan). Adenosine triphosphate (ATP), creatine phosphate (di-Tris), and phosphocreatine kinase were purchased from Sigma-Aldrich (St. Louis, MO). dATP, dGTP, dTTP, and dCTP were purchased from Toyobo (Osaka, Japan). Bovine serum albumin (BSA) was purchased from Seikagaku Corporation (Tokyo, Japan). AccuGel 19:1 (40%) for NER reactions and ProtoGel 30:0.8 (40%) for gel mobility shift assay were purchased from National Diagnostics (Atlanta, GA).
DNA, enzymes, and cell extract
The oligonucleotide containing Oz were prepared as described earlier. , The oligonucleotide containing 6-4PP were provided by Prof. Shigenori Iwai (Graduate School of Engineering Science, Osaka University). Purification and reconstitution of the XPC-RAD23B-His heterodimer were carried out as described earlier.  The whole cell extracts of a cell line from the patient with XP complementation group C (XP3BE) was prepared as described earlier.  The 32 P-labeled, blunt-ended 180 bp DNA fragments and internally 32 P-labeled, closed circular DNAs were prepared as described earlier [Figure 1]b. 
The detailed method was described earlier. , Briefly, the internally 32 P-labeled, double-stranded circular DNA substrates (1 × 10 5 cpm, ~29 ng) were incubated at 30°C for 1 h in 25 μL reactions containing 40 mM HEPES-KOH (pH 7.8); 5 mM MgCl 2 ; 0.5 mM DTT; 70 mM NaCl; 6.6% glycerol; 0.5 mM EDTA; 2 mM ATP; and 20 μM each of dATP, dGTP, and dTTP; 8 μM dCTP; 22.5 mM creatine phosphate (di-Tris); phosphocreatine kinase (1.25 μg); BSA (9 μg); the XP3BE whole cell extracts (100 μg protein); and the XPC-RAD23BH is complex (4 ng). The reactions were stopped by addition of EDTA to a final concentration of 10 mM, and DNA was purified and subjected to 10% denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Radioactivity was quantified using the BAS2500 bioimaging analyzer (Fujifilm, Tokyo, Japan). X-ray films (Fujifilm, RX-U) were exposed to the dried gel at -80°C with an intensifying screen.
Gel mobility shift assay for XPC-binding
The detailed method was described earlier. , Briefly, binding reactions (10 μL) were carried out at 30°C for 30 min in mixtures including 20 mM sodium phosphate (pH 7.4), 5 mM MgCl 2 , 1 mM EDTA, 150 mM NaCl, 1 mM DTT, 5% glycerol, 0.01% Triton X-100, BSA (1 μg), 32 P-labeled probe DNA (0.35 fmol), covalently closed circular plasmid DNA (1.5 ng), and the recombinant XPC-RAD23B-His complex (4 ng). The reactions were then chilled on ice. The mixtures were directly subjected to 4% nondenaturing PAGE.
| Results And Discussion|| |
Cell-free NER activity was investigated using the human whole cell extracts and covalently closed circular DNAs containing an internal 32 P-label 5' to the Oz lesion. As described earlier, , cell-free NER reactions were performed using the whole cell extracts lacking the XPC protein and supplemented with purified XPC-RAD23B. In the absence of XPC-RAD23B, the NER reaction did not occur [Figure 2].
|Figure 2: The NER incision of the duplex containing Oz compared with 6-4PP. The indicated internally labeled substrates were assayed for NER incision in the XP3BE whole cell extracts supplemented with (+) or without (-) XPC-RAD23B. The part of the autoradiograph showing the dual incision products is presented. "M" is the 32P-labeled 14mer|
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The dual incision activity on Oz opposite cytosine was detected. However, the efficiency was much lower than that for a 6-4PP [Figure 2]. By quantification, the efficiency of the NER incision on Oz:C was ~7% of that on 6-4PP. The structure of Oz is planar [Figure 1]a, and Oz is not a highly bulky lesion. Since efficient recognition by the NER incision system requires disrupted base pairs, , the reactivity on Oz was understandably low.
XPC-RAD23B heterodimer recognizes unstable base pairs , and initiates NER in vitro.  Therefore, to examine the damage-binding affinity of XPC-RAD23B, we used the gel mobility shift technique.  Blunt-ended radiolabeled DNA fragments (180 bp in length) containing a single lesion at a defined position were used for binding reactions with the purified recombinant human XPC-RAD23B complex. The percentages of the labeled DNA probes bound to XPC-RAD23B were calculated [Figure 3]. Using the probe containing 6-4PP (positive control), it was demonstrated that XPC-RAD23B is capable of binding 6-4PP in a highly specific manner. These results were consistent with the previous data. 
|Figure 3: XPC-RAD23B binding activity to Oz-containing oligonucleotides. The 32P-labeled DNA substrates (180 bp fragments) (0.35 fmol each) were incubated at 30°C for 30 min with XPC-RAD23B (4 ng). The percentage of the 32P-labeled DNA substrate bound to XPC-RAD23B was calculated. The mean values and standard errors were calculated from two independent experiments|
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The binding of Oz:C with XPC-RAD23B was lower than that of 6-4PP [Figure 3]. Given that the presence of bulky damage sites is crucial for recognition by XPC-RAD23B ,, and that Oz is a less bulky damage site than 6-4PP, the low binding activity on Oz seems reasonable. Thus, low incision reactivity on Oz in [Figure 2] can be accounted for by low binding of Oz with XPC-RAD23B.
| Conclusion And Implications|| |
It is unfortunate that the efficiency on the substrate containing Oz was much lower than that on 6-4PP in the cell-free NER reaction, owing to low binding of Oz with XPC-RAD23B. There might be an unknown factor required in vivo for efficient binding of XPC-RAD23B to Oz:C. We will continue to search for an efficient repair system in human cells that protects against G: C-C: G transversions by Oz by recognizing the Oz:C base pair.
This work was supported by a research grant from the Japan Prize Foundation. We also thank Prof. S. Iwai for providing the oligonucleotide containing 6-4PP.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]