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The spread of the novel coronavirus disease (COVID-19) has caused a global pandemic. Exiting agents that act on proteins including 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), helicase, RNA-dependent RNA polymerase (RdRp), spike glycoprotein (S protein), and others in similar viruses [1,2] are likely used as antiviral drugs against the novel coronavirus (SARS-CoV-2). Data on Chemical Abstract Service show that the potential drug candidates against 3CLpro and RdRp are more than those against other targets [2].

There is adequate evidence for the carcinogenicity of cadmium (Cd). However, a significant unaddressed question remains as to how this metal actually causes malignant transformation (tumor initiation). Since it has been shown that Cd only has the weak direct interaction potential with DNA, the metal is recognized as an indirect genotoxicant and mutagen. Currently, Cd-mediated "epigenetic" modifications, such as changes in DNA methylation resulting in alteration in target gene expression, coupled with cancer progression, are the focus of mechanistic research. We have reported that the apolipoprotein E (ApoE) gene, a suppressor of cell invasion, is an early Cd target, and is involved in the malignant transformation of TRL 1215 rodent liver cells. Cd exposure suppresses ApoE expression which can be re-activated with 5-aza-2'-deoxycytidine, a DNA demethylating agent. In the present study, we sought direct evidence of Cd-induced DNA hypermethylation of the ApoE promoter region by performing bisulfite sequencing and real-time quantitative methylation-specific PCR. Our data clearly suggest that Cd can down-regulate the expression of ApoE via introduction of excess DNA methylation in the promoter region of ApoE during malignant transformation of TRL 1215 cells.

Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.

TAL2 is a transcription factor required in the normal development of mouse brain. In a previous study, we demonstrated that the expression of Tal2 gene is induced by the complex of all-trans retinoic acid (atRA) and retinoic acid receptor α (RARα) in mouse embryonal carcinoma P19 cells. atRA is also known to be important in inducing P19 cells to differentiate into the neural lineage. Therefore, we believe that the function of TAL2 in neural differentiation may be clarified by utilizing P19 cells. As the atRA-RARα complex induced the expression of Tal2, we focused on the regulatory region that is involved in its transcription. The atRA-RARα complex occupies a characteristic retinoic acid response element (RARE) located in the promoter of target genes. Therefore, we searched for RARE on the mouse Tal2 and found that a RARE-like element was located in the intron. We also found that a TATA-box-like element was located in the 5'-region of Tal2. Involvement between transcriptional activity and the TATA-box-like element was confirmed in the luciferase assay, and TATA-box binding protein was bound to this element upstream of Tal2 in P19 cells. atRA signaling activated the transcription through the RARE-like element, and RARα was bound to this element on Tal2 in P19 cells. In addition, the interaction between these elements on Tal2 was shown in the chromatin immunoprecipitation assay. These results suggest that the transcription of Tal2 is coordinately mediated by two distal regulatory elements.

Guanine is the most readily oxidized of the four DNA bases, and guanine oxidation products cause G:C-T:A and G:C-C:G transversions through DNA replication. 8-Oxo-7,8-dihydroguanine (8-oxoG) causes G:C-T:A transversions but not G:C-C:G transversions, and is more readily oxidized than guanine. This review covers four major findings. (i) 2,2,4-Triamino-5(2H)-oxazolone (Oz) is produced from guanine and 8-oxoG under various oxidative conditions. Guanine is incorporated opposite Oz by DNA polymerases, except REV1. (ii) Several enzymes exhibit incision activity towards Oz. (iii) Since the redox potential of GG is lower than that of G, contiguous GG sequences are more readily oxidized by a one-electron oxidant than a single guanine, and OzOz is produced from GG in double-stranded DNA. Unlike most DNA polymerases, DNA polymerase ζ efficiently extends the primer up to full-length across OzOz. (iv) In quadruplex DNA, 3′-guanine is mainly damaged by one-electron oxidation in quadruplex DNA, and this damage depends on the highest occupied molecular orbital (HOMO). The oxidation products in quadruplex DNA are different from those in single-stranded or double-stranded DNA.

In a previous study, we showed that formylmethylflavin (FMF) can bind to cysteine. In this study, FMF was reacted with native peptides (CG and CKLVFF) containing an N-terminal cysteine. The formation of flavin-CG and flavin-CKLVFF was confirmed using HPLC and ESI-MS. Storage of flavin-CKLVFF in DMSO at −30 °C for 7 days resulted in no detectable deposition. In contrast, flavin-CKLVFF formed deposits when stored in water at −30 °C for 1 day, but no deposit was observed in the aqueous solution of flavin-CKLVFF after 7 days storage in the presence of 0.1% Triton X-100.

Guanine is the most easily oxidized among the four DNA bases, and some guanine-rich sequences can form quadruplex structures. In a previous study using 6-mer DNA d(TGGGGT), which is the shortest oligomer capable of forming quadruplex structures, we demonstrated that guanine oxidation products of quadruplex DNA differ from those of single-stranded DNA. Therefore, the hotooxidation products of double-stranded DNA (dsDNA) may also differ from that of quadruplex or single-stranded DNA, with the difference likely explaining the influence of DNA structures on guanine oxidation pathways. In this study, the guanine oxidation products of the dsDNA d(TGGGGT)/d(ACCCCA) were analyzed using HPLC and electrospray ionization-mass spectrometry (ESI-MS). As a result, the oxidation products in this dsDNA were identified as 2,5-diamino-4H-imidazol-4-one (Iz), 8-oxo-7,8-dihydroguanine (8oxoG), dehydroguanidinohydantoin (Ghox), and guanidinohydantoin (Gh). The major oxidation products in dsDNA were consistent with a combination of each major oxidation product observed in single-stranded and quadruplex DNA. We previously reported that the kinds of the oxidation products in single-stranded or quadruplex DNA depend on the ease of deprotonation of the guanine radical cation (G•+) at the N1 proton. Similarly, this mechanism was also involved in dsDNA. Deprotonation in dsDNA is easier than in quadruplex DNA and more difficult in single-stranded DNA, which can explain the formation of the four oxidation products in dsDNA.

The nucleobase derivative, 2,2,4-triamino-5(2H)-oxazolone (Oz), is an oxidation product of guanine or of 8-oxo-7,8-dihydroguanine that causes G-to-C transversions in DNA. Human NEIL1 (hNEIL1) and NTH1 (hNTH1) are homologues of two prokaryotic base excision repair enzymes, FPG/NEI and NTH, respectively. Here, we demonstrated that hNEIL1 and hNTH1 cleave Oz sites as efficiently as 5-hydroxyuracil sites. Thus, hNEIL1 and hNTH1 can repair Oz lesions. Furthermore, the nicking activities of these enzymes are largely independent of nucleobases opposite Oz; this finding indicates that removing Oz from Oz:G and Oz:A base pairs might cause an increase in the rate of point mutations in human cells.

DNA is constantly exposed to endogenous and exogenous oxidative stresses. Damaged DNA can cause mutations, which may increase the risk of developing cancer and other diseases. G:C-C:G transversions are caused by various oxidative stresses. 2,2,4-Triamino-5(2H)-oxazolone (Oz), guanidinohydantoin (Gh)/iminoallantoin (Ia) and spiro-imino-dihydantoin (Sp) are known products of oxidative guanine damage. These damaged bases can base pair with guanine and cause G:C-C:G transversions. In this study, the stabilization energies of these bases paired with guanine were calculated in vacuo and in water. The calculated stabilization energies of the Ia:G base pairs were similar to that of the native C:G base pair, and both bases pairs have three hydrogen bonds. By contrast, the calculated stabilization energies of Gh:G, which form two hydrogen bonds, were lower than the Ia:G base pairs, suggesting that the stabilization energy depends on the number of hydrogen bonds. In addition, the Sp:G base pairs were less stable than the Ia:G base pairs. Furthermore, calculations showed that the Oz:G base pairs were less stable than the Ia:G, Gh:G and Sp:G base pairs, even though experimental results showed that incorporation of guanine opposite Oz is more efficient than that opposite Gh/Ia and Sp.

Ischemia-reperfusion or traumatic brain injury induces the accumulation of reactive oxygen species (ROS) in brain. ROS causes oxidative stress to astrocytes as well as neurons and oxidative stress induces the damage to organelles, proteins or lipids. The removal of damaged cellular cytosolic components is indispensable for the cell to keep the homeostasis. Macroautophagy (hereafter referred to as autophagy) is the process to degrade defective proteins and damaged organelles. In the process of autophagy, damaged cellular cytosolic components are isolated by autophagosomes. Microtubule-associated protein 1 light chain 3B (LC3B) plays a significant role in the autophagosome formation, and the conversion from unconjugated-LC3B (LC3B-I) to phosphatidylethanolamine (PE) conjugated-LC3B (LC3B-II) is the index of the activitation of autophagy. GABARAPL1 is the paralogue of LC3B and the function of GABARAPL1 is not fully understood. In this study, we demonstrated GABARAPL1 mRNA and protein expression are up-regulated by H2O2 in rat C6 glioma cells and the induction of GABARAPL1 by H2O2 was accompanied with the conversion from LC3B-I to LC3B-II, indicating the formation of autophagosomes. Thus, GABARAPL1 may play a role in autophagic process, which is induced by H2O2. However, elucidation of the function of GABARAPL1 in autophagy will require further studies.

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.

Photosensitized oxidation of guanine provides various oxidation products, including 8-oxoguanine (8-oxoG) and imidazolone. Riboflavin (vitamin B2) is known as an effective photosensitizer for oxidation of guanine. Here, we show the user-friendly synthesis and photoreaction of a flavin-linked oligonucleotide; the practical synthesis of a hydroxyethyl-flavin (2) from commercially available riboflavin; and the preparation of a flavin-linked oligonucleotide using the phosphoramidite of 2. To demonstrate the usefulness of this method, the flavin-linked oligomer flavin-5′-d(T6CCT6)-3′ was synthesized. Flavin-5′-d(T6CCT6)-3′ and its complementary 5′-d(A6G8-oxoGA6)-3′ were irradiated under UV light (366 nm) at neutral pH. Enzymatic digestion of the irradiated mixture with P1 nuclease and alkaline phosphatase indicated that the 8-oxoG residue was oxidized to imidazolone. These results demonstrate that 8-oxoG is effectively oxidized to imidazolone by photosensitization of the terminal flavin via a hole-transfer mechanism, and imidazolone is formed by one-electron oxidation of 8-oxoG at neutral pH.

Mutations induced by oxidative DNA damage can cause diseases such as cancer. In particular, G:C-T:A and G:C-C:G transversions are caused by oxidized guanine and have been observed in the p53 and K-ras genes. We focused on an oxidized form of guanine, 2,2,4-triamino-5(2H)-oxazolone (Oz), as a cause of G:C-C:G transversions based on our earlier elucidation that DNA polymerases (Pols) α, β, γ, ε, η, I, and IV incorporate dGTP opposite Oz. The nucleotide insertion and extension of Pols δ, ζ, ι, κ, and REV1, belonging to the B- and Y-families of DNA polymerases, were analyzed for the first time. Pol δ incorporated dGTP, in common with other replicative DNA polymerases. Pol ζ incorporated dGTP and dATP, and the efficiency of elongation up to full-length beyond Oz was almost the same as that beyond G. Although nucleotide incorporation by Pols ι or κ was also error-prone, they did not extend the primer. On the other hand, the polymerase REV1 predominantly incorporated dCTP opposite Oz more efficiently than opposite 8-oxo-7,8-dihydroguanine, guanidinohydantoin, or tetrahydrofuran. Here, we demonstrate that Pol ζ can efficiently replicate DNA containing Oz and that REV1 can prevent G:C-C:G transversions caused by Oz.

Many oxidizing agents induce G-C to T-A and G-C to C-G transversions, and the frequency largely depends on the oxidative conditions. Guanine is the most oxidizable base among natural bases. The typical oxidative lesion product 8-oxoguanine (8-oxoG) is responsible for G-C to T-A transversion but not for G-C to C-G transversion, and 8-oxoG is more readily oxidized than guanine because of its lowered ionization potential. Recently, imidazolone (Iz), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) have been demonstrated as oxidative lesion products of guanine and 8-oxoG, which could be responsible for G-C to C-G transversions by forming specific base pair formations.

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. Although this residue is a potentially mutagenic lesion and is removed by several base excision repair enzymes, it is unknown whether fU is the substrate of nucleotide excision repair (NER). Here, we analyzed the binding specificity of XPC-HR23B, which initiates NER, and cell-free NER activity on fU opposite four different bases. The result of the gel mobility shift assay showed that XPC-HR23B binds the fU-containing substrates in the following order: fU:C >> fU:T > fU:G > fU:A. Furthermore, in the presence of XPC-HR23B, the dual incision activity was the same as the order of the binding affinity of XPC-HR23B to fU. Therefore, it is concluded that even fU, regarded as a shape mimic of thymine, can be recognized as a substrate of NER incision, and the efficiency depends on instability of the base pair.

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The present authors reported previously that MutM, Nth and Nei in Escherichia coli removed 5-foU from DNA. The present study identified 5-foU DNA glycosylases in Saccharomyces cerevisiae in order to clarify the repair mechanisms of 5-foU in eukaryotic cells.The borohydride-trapping assay and DNA-nicking assay were carried out to detect and characterize the repair activities for 5-foU in extracts from S. cerevisiae with oligonucleotides containing 5-foU at specific sites.Two proteins in crude extracts from S. cerevisiae formed covalent complexes with oligonucleotides containing site-specific 5-foU in the presence of NaBH4. Extracts from S. cerevisiae strains defective in either the NTG1 or the NTG2 gene lacked either one or the other of these two proteins. Purified Ntg1 and Ntg2 were trapped in such complexes by the 5-foU-containing oligonucleotides in the presence of NaBH4. Furthermore, purified Ntg1 and Ntg2 efficiently cleaved the oligonucleotide at the 5-foU site.The results indicate that both Ntg1 and Ntg2 are involved in the repair of 5-foU in DNA, and thereby serve to reduce mutations in S. cerevisiae.

Replacement of the furanose moiety of DNA with a cyclopentane ring produces a modified sugar analogue: Carbocyclic nucleic acid (CarNA). UV melting-temperature experiments demonstrate that the incorporation of 2'-deoxycarbaguanosine ((c)G) and 2'-deoxyaristeromycin ((c)A) of carbocyclic nucleosides into a DNA strand increases the stability of the CarNA/RNA hybrid. Circular Dichroism (CD) study indicates that the CarNA/RNA hybrid adopts an A-like conformation. To elucidate the molecular basis of the increased stability of the CarNA/RNA, the conformation of (c)G and (c)A were examined by (1)H NMR conformational analysis of (3)J(HH) coupling constants and ab initio molecular orbital (MO) calculations. These results show that the populations of N-type of (c)G and (c)A are higher than those of dG and dA, respectively, at different temperatures [For example, 37% (N%) of (c)G vs. 28%of dG, 36% (N%) of (c)A vs. 25% of dA at 278 K], which suggest that the cyclopentane rings of (c)G and (c)A prefer the N-type conformation in two-state N-S pseudorotional equilibrium in comparison with the furanose rings of dG and dA. The DeltaH degrees of (c)G (DeltaH degrees = - 0.43 kcal mol(-1)) and (c)A (DeltaH degrees = - 0.41kcal mol(-1)) are lower than that of dG (dG = - 1.8 kcal mol(-1)) and dA (dA = - 1.0 kcal mol(-1)), respectively, which suggest that the gauche effect in the (c)A and (c)G driving N-S pseudorotional equilibrium to S-type is reduced by replacement of the 4'-oxygen by a CH(2) group. These results suggest that the preferred N-type of the (c)G and (c)A leads to the A-like conformation, which contributes to the stability of CarNA/RNA hybrid.

The nucleoside 2,2,4-triamino-5(2H)-oxazolone (Oz) can result from oxidative damage to guanine residues in DNA. Despite differences among the three polymerases (Polβ, KF exo−, and Polη) regarding nucleotide incorporation patterns opposite Oz, all three polymerases can incorporate guanine opposite Oz. Based onab initiocalculations, we proposed a structure for a stable Oz:G base pair. Here, to assess the stability of each Oz-containing base pair (Oz:G, Oz:A, Oz:C, and Oz:T) upon DNA replication, we determined the efficiency of Polβ-, KF exo−-, or Polη-catalyzed primer extension beyond each base pair. With each polymerase, extension beyond Oz:G was more efficient than that beyond Oz:A, Oz:C, or Oz:T. Moreover, thermal denaturation studies revealed that theTmvalue for the duplex containing Oz:G was significantly higher than those obtained for duplexes containing Oz:A, Oz:C, or Oz:T. Therefore, the results fromab initiocalculations along with those from DNA replication assays and thermal denaturation experiments supported the conclusion that Oz:G is the most stable of the Oz-containing base pairs.

DNA is constantly being oxidized, and oxidized DNA is prone to mutation, moreover, guanine is highly sensitive to several oxidative stressors. Several oxidatively damaged forms of guanine—including 2,2,4-triamino-5(2H)-oxazolone (Oz), iminoallantoin (Ia), and spiroiminodihydantoin (Sp)—can be paired with guanine, and cause G:C-C:G transversions. Previous findings indicate that guanine is incorporated more efficiently opposite Oz than opposite Ia or Sp, and that these differences in efficiency cannot be explained by differences in the stabilities of G:Oz, G:Ia, and G:Sp base pairs calculated ab initio. Here, to explain previous experimental result, we used a 3-base-pair model DNA duplex to calculate the difference in the stability and the distortion of DNA containing a G:Oz, G:Ia, or G:Sp base pair. We found that the stability of the structure containing 5ꞌ and 3ꞌ base pairs adjacent to G:Oz was more stable than that containing the respective base pairs adjacent to G:Ia or G:Sp. Moreover, the distortion of the structure in the DNA model duplex that contained a G:Oz was smaller than that containing a G:Ia or G:Sp. Therefore, our discussion can explain the previous results involving translesion synthesis past an oxidatively damaged guanine.

TAL2 is a member of the basic helix-loop-helix family and is essential for the normal development of the mouse brain. However, the function of TAL2 during brain development is unclear. P19 cells are pluripotent mouse embryonal carcinoma cells that adopt neural fates upon exposure to all-trans retinoic acid (atRA) and culture in suspension. We found that the expression of Tal2 gene was induced in P19 cells after addition of atRA in suspension culture. Tal2 expression was detected within 3 h after the induction and had nearly returned to basal levels by 24 h. When GFP-tagged TAL2 (GFP-TAL2) was expressed in P19 cells, we observed GFP-TAL2 in the nucleus. Moreover, we showed that atRA and retinoic acid receptor α regulated Tal2 expression. These results demonstrate for the first time that atRA induces Tal2 expression in P19 cells and suggest that TAL2 commits to the acquisition of neural fate in brain development.

Background: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G·C→T·A and G·C→C·G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G·C→T·A transversion mutations. Although the number of G·C→C·G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G·C→C·G transversions is not known. Results: To determine which oxidative products are responsible for G·C→C·G transversion mutations, we photooxidized 5′-d(AAAAAAGGAAAAAA)/5′-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4°C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2′-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-β-D- erythro -pentofuranosyl)amino]-4 H -imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4 H -imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz·G base pairs are formed. The ‘reverse' approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz·G base pair. Conclusions: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz·G base pair formation can explain the G·C→C·G transversion mutations that appear under oxidative conditions.

5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of5-foU in vivo.The modified base was incorporated site-specifically into the recognition site of restriction endonuclease SalI (5'-GTCGAC) or AflII (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli.When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A--C:G and T:A--A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids.The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.

DNA is damaged by various oxidative stresses. Oxidized DNA can increase mutations and the risk of cancer. Guanine is highly sensitive to several oxidative stresses due to its low oxidation potential. It is known that 2,2,4-triamino-5(2H)-oxazolone (Oz), iminoallantoin (Ia) and spiroiminodihydantoin (Sp) are oxidized guanine lesions. These oxidized bases can be paired with guanine and cause G:C-C:G transversions. Although our previous results showed more effective incorporation of guanine opposite Oz compared to that opposite Ia or Sp (K. Kino et al., ChemBioChem, 2009), Oz:G base pair was less stable than Ia:G or Sp:G base pair by ab initio calculation (M. Suzuki et al., Molecules, 2012). Oz:G forms two hydrogen bonds and is planar, while Ia:G and Sp:G have three hydrogen bonds and are nonplanar. This difference can be due to the fact that Ia:G and Sp:G contain a sp3 carbon but not Oz:G. Since stacking planar base pairs in DNA significantly enhances the stability of DNA duplex, we investigate whether the stability of DNA duplex containing the oxidized lesion can be accounted for by the stacking effect in this study. The structure of DNA duplex containing Oz:G was the most stable. This our data agreed with the previous results that translesion synthesis across Oz was more effective than Ia or Sp.

The deposition of Amyloid beta peptides (Aβ) in the formation of amyloid fibrils is believed to be causally linked to Alzheimer's disease. A short Aβ fragment (KLVFF; Aβ16-20) can bind full-length Aβ. The Aβ recognition peptide including KLVFF was attached with hydrophilic groups, and then the product altered the Aβ aggregation pathways and inhibited Aβ toxicity (Tjernberg, L. O. et al. J. Biol. Chem. 1996, 271, 8545-8548. ). Since flavins is widely known as biological oxidation reagents, the flavin-linked KLVFF is likely to directly hydroxylate aggregated Aβ fibrils and then disrupt the aggregated Aβ fibril. 2-Aminoethanethiol derivatives, such as cysteine, is reacted with aldehydes, and five-membered heterocyclic ring is formed via formation of imine. Since formylmethylflavin (FMF) contains aldehyde group, FMF is likely to bind cysteine. Then, the peptides containing Aβ recognition region and cysteine, such as CKLVFF, also can be attached with FMF to form the flavin-linked KLVFF. The preliminary experiment of this reaction, the reaction between FMF and cysteine, was investigated. FMF (2.84 mg, 10 μmol) was suspended in phosphate buffer (pH 7, 10 ml), and cysteine (2.42 mg, 20 μmol) was added. The suspension was stirred at 65oC for 1h. As a result, FMF was completely reacted, and flavin-linked cystein were detected using HPLC and ESI-MS.

5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood. In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E. coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites. The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides. Extracts from strains defective in thenth, nei, or mutM gene lacked one of the proteins. All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant. The introduction of a plasmid carrying the nth,nei, or mutM gene into the E. colitriple mutant restored the formation of the corresponding protein-DNA complex. Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH4. Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site. In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E. coli. The mutation frequency of the plasmid was significantly increased in theE. coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains. From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E. coli.

κ-Casein (CSN3) is known to play an essential role in controlling the stability of the milk micelles. We found that the expression of Csn3 was induced by all-trans retinoic acid (ATRA) during neural differentiation in P19 embryonal carcinoma cells from our study using DNA microarray. In this paper, we describe the detailed time course of Csn3 expression and the induction mechanism of Csn3 transcription activation in this process. The Csn3 expression was induced rapidly and transiently within 24 h of ATRA treatment. Retinoic acid receptor (RAR)-specific agonists were used in expression analysis to identify the RAR subtype involved upregulation of Csn3; a RARα-specific agonist mimicked the effects of ATRA on induction of Csn3 expression. Therefore, RARα may be the RAR subtype mediating the effects of ATRA on the induction of Csn3 gene transcription in this differentiation-promoting process of P19 cells. We found that the promoter region of Csn3 contained a typical consensus retinoic acid response element (RARE), and this RARE was necessary for ATRA-dependent transcriptional regulation. We confirmed that RARα bound to this RARE sequence in P19 cells. These findings indicated that the Csn3 expression is upregulated via ATRA-bound RARα and binding of this receptor to the RARE in the Csn3 promoter region. This will certainly serve as a first step forward unraveling the mysteries of induction of Csn3 in the process of neural differentiation.

A new method for the synthesis of 5-foU-containing oligonuleotides by phosphoramidite chemistry and subsequent post-oxidation with sodium periodate was developed. 5-(1,2-Dihydroxyethyl)uracil-containing oligomers, which are readily converted to 5-foU-containing oligomers by sodium periodate oxidation, were synthesized according to the standard β-cyanoethyl phosphoramidite chemistry. The phosphoramidite of a protected 5-(1,2-dihydroxyethyl)-2′-deoxyuridine derivative was prepared from 5-iodo-2′-deoxyuridine in 7 steps. UV melting behavior of these three oligomers demonstrated that the 5-foU-A base pair are less stable than the T-A base pair.

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.

8-Oxo-7,8-dihydroguanine was specifically oxidized by iodine with aqueous KI. Under acidic conditions, the major product was dehydro-guanidinohydantoin. Under basic conditions, two diastereoisomers of spirohydantoin were chiefly obtained. In addition, unstable diimine was detected for the first time.

8-Oxoguanine is the typical oxidative product, but 8-oxoguanine is further oxidized by several oxidizing agents. Here, we investigated the oxidation of 2'-deoxy-8-oxoguanosine with aqueous iodine.

Nuclear factor-kappaB (NF-kappaB) has been considered as a good target for the treatment of many diseases. Although a lot of NF-kappaB inhibitors have already been reported, many of them have several common problems. Thus, we attempted to identify novel NF-kappaB inhibitors to be unique lead compounds for creating new pharmaceuticals. In the present study, we screened our chemical library for compounds that directly inhibit the DNA binding of NF-kappaB by using fluorescence correlation spectroscopy (FCS). Consequently, we identified a promising compound, 4,6-dichloro-N-phenyl-1,3,5-triazin-2-amine, referred to as NI241. It mediated a dose-dependent inhibition of the DNA binding of NF-kappaB p50. Its analogues also showed dose-dependent inhibition and their inhibitory effects were altered by the substituents on the N-phenyl group. Furthermore, we predicted the binding mode of NI241 with p50 in silico. In this model, NI241 forms three hydrogen bonds with Tyr60, His144, and Asp242 on p50, which are important amino acid residues for the interaction with DNA. These results suggest that NI241 with structural novelty may serve as a useful scaffold for the creation of new NF-kappaB inhibitors by rational optimization.

Photoirradiation in the presence of riboflavin led to guanine oxidation and the formation of imidazolone. Meanwhile, riboflavin itself was degraded by ultraviolet light A (UV-A) and visible light (VIS) radiation, and the end product was lumichrome. VIS radiation in the presence of riboflavin oxidized guanine similarly to UV-A radiation. Although UV-A radiation with lumichrome oxidized guanine, VIS radiation with lumichrome did not. Thus, UV-A radiation with riboflavin can oxidize guanine even if riboflavin is degraded to lumichrome. In contrast, following VIS radiation degradation of riboflavin to lumichrome, VIS radiation with riboflavin is hardly capable of oxidizing guanine. The consequences of riboflavin degradation and guanine photooxidation can be extended to flavin mononucleotide and flavin adenine dinucleotide. In addition, we report advanced synthesis; carboxymethylflavin was obtained by oxidation of formylmethylflavin with chlorite and hydrogen peroxide; lumichrome was obtained by heating of formylmethylflavin in 50% AcOH; lumiflavin was obtained by incubation of formylmethylflavin in 2 M NaOH, followed by isolation by step-by-step concentration.

A DNA-linked polymer micelle was prepared through a phase transition of an amphiphilic copolymer consisting of single-stranded DNA as the hydrophilic part and thermo-responsive poly(N-isopropylacryl-amide) as the hydrophobic part. Duplex formation on the surface of micelle decreased the colloidal stability, whereas a terminal single-base mismatch at the interface between the DNA shell and the disperse medium increased the colloidal stability. Based on this change, we successfully detected base pairing of 7,8-dihydro-8-oxo-2'-deoxyguanine with adenine. This method will be useful for discovering a Watson-Crick or non Watson-Crick base pair of an unknown oxidative-damaged base with each four canonical one.

The oxidation and deamination of 5-methylcytosine (5mC) in DNA generates a base-pair between 5-hydroxymethyluracil (5hmU) and guanine. 5hmU normally forms a base-pair with adenine. Therefore, the conversion of 5mC to 5hmU is a potential pathway for the generation of 5mC to T transitions. Mammalian cells have high levels of activity of 5hmU-DNA glycosylase, which excises 5hmU from DNA. However, glycosylases that similarly excise 5hmU have not been observed in yeast or Escherichia coli. Recently, we found that E.coli MutM, Nei and Nth have DNA glycosylase activity for 5-formyluracil, which is another type of oxidation product of the thymine methyl group. In this study, we examined whether or not E.coli MutM, Nei and Nth have also DNA glycosylase activity that acts on 5hmU in vitro. When incubated with synthetic duplex oligonucleotides containing 5hmU:G or 5hmU:A, purified MutM, Nei and Nth cleaved the 5hmU:G oligonucleotide 58, 5 and 37 times, respectively, more efficiently than the 5hmU:A oligonucleotide. In E.coli, the 5hmU-DNA glycosylase activities of MutM, Nei and Nth may play critical roles in the repair of 5hmU:G mispairs to avoid 5mC to T transitions.

5-formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. The elucidation of repair mechanisms for 5-foU will yield important insights into the biological consequences of the lesion. Recently, we reported that 5-foU is recognized and removed from DNA by Escherichia coli enzymes Nth (endonuclease III), Nei (endonuclease VIII) and MutM (formamidopyrimidine DNA glycosylase). Human cells have been shown to have enzymatic activities that release 5-foU from X-ray-irradiated DNA, but the molecular identities of these activities are not yet known. In this study, we demonstrate that human hNTH1 (endonuclease III homolog) has a DNA glycosylase/AP lyase activity that recognizes 5-foU in DNA and removes it. hNTH1 cleaved 5-foU-containing duplex oligonucleotides via a beta-elimination reaction. It formed Schiff base intermediates with 5-foU-containing oligonucleotides. Furthermore, hNTH1 cleaved duplex oligonucleotides containing all of the 5-foU/N pairs (N = G, A, T or C). The specific activities of hNTH1 for cleavage of oligonucleotides containing 5-foU and thymine glycol were 0.011 and 0.045 nM/min/ng protein, respectively. These results indicate that hNTH1 has DNA glycosylase activity with the potential to recognize 5-foU in DNA and remove it in human cells.

The TET-family proteins (TAF15, EWS and TLS) are the RNA binding proteins involved in multiple levels of cellular functions. The RNA binding domain of those proteins is known as the important region for cellular functions. But little is known about the RNA binding specificity of TET-family proteins. In order to investigate the RNA binding properties of the TET-family proteins, we performed electrophoretic mobility shift assay using recombinant Flag-tagged TLS and guanine-rich and RNAs. It was found that TLS binds to human telomeric RNA in the presence of KCl, but not in the presence of LiCl.

The formation of quadruplex structure changed the site reactivity and the kinds of guanine photooxidation products of d(TGGGGT). In quadruplex DNA, 8-oxo-7,8-dihydroguanine (8oxoG) and dehydroguanidinohydantoin (Ghox) were mainly formed, although 2,5-diamino-4H-imidazol-4-one (Iz) was mainly formed in single-stranded DNA. In addition, 3′-guanine was specifically oxidized in quadruplex DNA compared with single-stranded DNA, which depended on the localization of the HOMO.

of d(A6GGA6)/d(T6CCT6) with sensitizer under different UV irradiation periods was examined. Enzymatic digestion of photolysate revealed that the amount of 8-oxoG initially increased with decrease of G and then decreased with the formation of Iz. In order to investigate whether Iz was formed by one electron oxidation of 8-oxoG through long-range hole migration, photoirradiation of anthra- quinone-linked oligomer was examined. The results clearly demonstrated that 8- oxoG was efficiently converted to Iz under irradiation conditions. These results suggest that Iz generated from 8-oxoG induces GC->CG transversion under oxidative condition. INTRODUCTIONgenome is constantly assaulted by oxidative stress, which causes cancer and aging. The reactive species are potent genotoxins which attack DNA and generate oxidative lesions. Therefore, considerable effort has focused upon understanding the mechanisms of oxidative DNA damage. In genome, guanine (G) is highly susceptible to oxidative stress due to the lowest redox potential. Atypical lesion is 7,8- dihydro-8-oxo-2' -deoxyguanosine (8-oxoG), which is formed under various oxidative conditions (Fig. la). Thus, the biological impact of 8-oxoG has been extensively investigated. Since 8-oxoG can pair with (a)

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3′-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5′-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K+ and d(TAGGGT)4T +2K+ localized at the 5′-guanine, and that the HOMO shifted from the 5′-guanine to the 3′-guanine by the addition of a 5′-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K+ and d(TAGGGT)4T +2K+ matched the experimental results and suggest that d(TGGGGT)4 contains a 5′-capping cation in solution.

Introduction 2,2,4-Triamino-5(2H)-oxazolone (Oz) in a DNA strand is an oxidation product of guanine and 8-oxo-7, 8-dihydroguanine, and such a lesion can cause G-to-C transversions. Previously, Fpg/Nei and Nth were shown to have incision activity on Oz. Findings We investigated the activities of chlorella virus pyrimidine dimer glycosylase (cvPDG) and Escherichia coli endonucleases IV (Nfo) and V (Nfi) on Oz. Although the three enzymes have different repair mechanisms from Fpg/Nei and Nth, they still had incision activity on Oz. Conclusions Given the incision activities of cvPDG, Nfo and Nfi on Oz in addition to Fpg/Nei and Nth, Oz is DNA damage that can be repaired by diverse enzymes.