Katsuhito Kino

New synthetic method of 5-formyluracil-containing oligonucleotides and their melting behavior

Abstract 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.

Sequence Dependence of Fluorescence Emission and Quenching of Doubly Thiazole Orange Labeled DNA: Effective Design of a Hybridization-Sensitive Probe

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.

Identification of repair enzymes for 5-formyluracil in DNA. Nth, Nei, and MutM proteins of Escherichia coli.

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.

Identification of Ewing’s sarcoma protein as a G-quadruplex DNA- and RNA-binding protein

The Ewing’s sarcoma (EWS) oncogene contains an N‐terminal transcription activation domain and a C‐terminal RNA‐binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg‐Gly‐Gly (RGG) domain of the C‐terminal in EWS binds to the G‐rich single‐stranded DNA and RNA fold in the G‐quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration‐dependent manner by the formation of a stabilized G‐quadruplex DNA–RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg‐Gly‐Gly sites and RGG containing Arg methylated by protein arginine N‐methyltransferase 3 decrease the binding ability of EWS to G‐quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G‐quadruplex DNA and RNA via the Arg residues in it.

Replication in vitro and cleavage by restriction endonuclease of 5-formyluracil- and 5-hydroxymethyluracil-containing oligonucleotides

PURPOSE To investigate the biological consequences of 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). MATERIALS AND METHOD The authors constructed 22-mer oligonucleotides containing a 5-foU or 5-hmU residue at the same sites. The effects of such modifications on the ability to serve as a template for DNA polymerase and on the cleavage by sequence-specific restriction endonuclease were examined. RESULTS The Klenow fragment of DNA polymerase I and Thermus thermophilus DNA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5-FoU directed the incorporation of dCMP in addition to dAMP opposite the lesion during DNA synthesis. The DNA polymerases incorporated only dAMP opposite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and SalI inhibited or prevented the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-containing oligonucleotides at the same rate as the T-containing oligonucleotides. CONCLUSIONS These results indicated that the 5-foU-A base pair is less stable than the T-A base pair and that 5-foU can form a base pair with C in addition to A. It was also demonstrated that the oxidation of thymine to 5-hmU does not result in substantial deterioration.

Eukaryotic DNA Polymerases α, β and ε Incorporate Guanine Opposite 2,2,4-Triamino-5(2H)-oxazolone

Alterations of genetic information by endogenous and exogenous oxidative stress have been implicated in carcinogenesis, aging, neurological syndromes, and the process of inflammation. Considerable efforts have thus focused on understanding the molecular mechanisms of DNA damage and repair. Oxidatively damaged DNA is particularly prevalent at guanine bases, at which reactive oxygen or nitrogen species, as well as hole transfer, cause >20 lesions at guanine. Various oxidative stresses lead to G:C–T:A and G:C–C:G transversions; this suggests a close relationship between frequent mutations of guanine and its higher susceptibility to oxidation, compared to that of other bases. Adenine incorporation opposite sites of oxidative guanine damage, which occurs through the action of polymerases, is regarded as a premutagenic lesion and causes G:C–T:A transversions, while guanine incorporation opposite such lesions initiates G:C–C:G transversions. Both 8-oxo-7,8-dihydroguanine (8oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) are known as ubiquitous markers of oxidatively damaged DNA. Eukaryotic polymerases incorporate adenine but not guanine opposite 8oxoG lesions; that is, 8oxoG:A base pairs cause G:C–T:A transversions. 6] Opposite FapyG lesions, adenine is also incorporated more efficiently than guanine; this indicates G:C–C:G transversions to be the result of other oxidative factors. Although guanidinohydantoin (Gh) and 2,5-diamino-4Himidazol-4-one 9] (Iz, Figure 1 A) predominantly induce G:C– C:G transversions in E. coli, there is no evidence that these lesions are premutagenic lesions that cause G:C–C:G transversions through the action of eukaryotic DNA polymerase. Iz can be slowly hydrolyzed to 2,2,4-triamino-5ACHTUNGTRENNUNG(2 H)-oxazolone (Oz) under physiological conditions; 11] this produces an equilibrium between the closedand open-ring structures (Figure 1 A). In liver DNA, two to six molecules of Oz are found per 10 guanines. Oz also appears to cause G:C–T:A transversions through the action of E. coli polymerase as well as in E. coli. Therefore, we examined Gh, Iz and Oz to determine the mechanism of point mutations resulting from oxidized lesions through the action of eukaryotic DNA polymerases. Human DNA polymerase a (hPol a) and rat DNA polymerase b (rPol b) were used for in vitro primer extension analysis of template oligonucleotides containing site-specific Gh or Iz (Figure 1 B) to determine nucleotide insertions opposite each lesion. Human Pol a and rPol b incorporated guanine and adenine opposite Gh, and guanine and cytosine opposite Iz (lanes 14–17,19–22 in Figure 1 C, D); this confirmed that both enzymes induce premutagenic states with Gh leading to G:C–T:A and G:C–C:G transversions and Iz leading to G:C–C:G transversions through the action of mammalian DNA polymerases a and b. We next investigated nucleotide insertion opposite Oz. Interestingly, guanine was almost exclusively inserted by hPol a and rPol b (lanes 9–12 in Figure 1 C, D); similar results were also obtained by using calf thymus polymerase a and human polymerase b (Figure S1). The kinetic parameters of guanine insertion by mouse polymerase a (mPol a) and rPol b are summarized in Figure S2. The calculated relative frequency of G misinsertion was substantially higher opposite Oz (1.7 10 5 in mPol a, 3.6 10 6 in rPol b) than opposite Gh (7.5 10 7 in mPol a, 5.9 10 7 in rPol b). In addition, full-length elongation of the primer was more efficient across Oz than across Gh (Figure 2 A, B); this suggests a higher potential of Oz than Gh to cause G:C–C:G transversions through the action of mammalian DNA polymerases a and b. To gain more insight into G:C–C:G transversions resulting from Oz, we investigated nucleotide insertions by human polymerase g (hPol g) and Saccharomyces cerevisiae polymerase e (scPol e), which both possess 3’!5’ exonuclease activity. This activity produced significant amounts of degraded products of the 5’-P-labeled primers; only part of the autoradiograms showing extended primer bands are presented in Figure 3 (whole gel images are presented in Figures. S3 and S4). Unlike in the case of the Gh template, hPol g appeared to insert both guanine and adenine opposite Oz, with the former being more [a] Dr. K. Kino, Prof. Dr. H. Miyazawa Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa 769-2193 (Japan) Fax: (+ 81) 87-894-5111 E-mail : kkino@kph.bunri-u.ac.jp [b] Dr. K. Kino, Prof. Dr. K. Sugasawa, Dr. T. Mizuno, Prof. Dr. F. Hanaoka Cellular Physiology Laboratory, RIKEN, Saitama 351-0198 (Japan) [c] Prof. Dr. K. Sugasawa Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Hyogo 657-8501 (Japan) [d] Dr. T. Bando, Prof. Dr. H. Sugiyama Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan) [e] Dr. M. Akita, Prof. Dr. F. Hanaoka Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871 (Japan) [f] Prof. Dr. F. Hanaoka Faculty of Science, Gakushuin University, Tokyo 171-8588 (Japan) Fax: (+ 81) 3-5992-1029 E-mail : fumio.hanaoka@gakushuin.ac.jp [g] Prof. Dr. K. Sugasawa, Dr. T. Mizuno, Prof. Dr. F. Hanaoka SORST (Japan) Science and Technology Agency (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900492.

Photoirradiation products of flavin derivatives, and the effects of photooxidation on guanine

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.

G-Quadruplex DNA- and RNA-Specific-Binding Proteins Engineered from the RGG Domain of TLS/FUS

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.

A DNA oligomer containing 2,2,4-triamino-5(2H)-oxazolone is incised by human NEIL1 and NTH1.

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.

Analysis of Guanine Oxidation Products in Double-Stranded DNA and Proposed Guanine Oxidation Pathways in Single-Stranded, Double-Stranded or Quadruplex DNA

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.