Search

Fat mass obesity-associated protein (FTO) is a DNA/RNA demethylase involved in the epigenetic regulation of various genes and is considered a therapeutic target for obesity, cancer, and neurological disorders. Here, we aimed to design novel FTO-selective inhibitors by merging fragments of previously reported FTO inhibitors. Among the synthesized analogues, compound 11b, which merges key fragments of Hz (3) and MA (4), inhibited FTO selectively over alkylation repair homologue 5 (ALKBH5), another DNA/RNA demethylase. Treatment of acute monocytic leukemia NOMO-1 cells with a prodrug of 11b decreased the viability of acute monocytic leukemia cells, increased the level of the FTO substrate N6-methyladenosine in mRNA, and induced upregulation of MYC and downregulation of RARA, which are FTO target genes. Thus, Hz (3)/MA (4) hybrid analogues represent an entry into a new class of FTO-selective inhibitors.

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT–histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3–H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid–H2A steric collision on the H2A-docking surface of the H3–H4 tetramer within the nucleosome induces H2A–H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2′-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2′ position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5′- and 3′-protected 2′-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2′-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme–DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.

Cancer chemoprevention is the use of chemical agents to inhibit, delay or reverse carcinogenesis. We established a novel method to evaluate agents for use in the chemoprevention of reactive oxygen species (ROS)-associated cancer. Induction of renal cell carcinoma in rats by ferric nitrilotriacetate (Fe-NTA) is an established model of ROS-associated cancer. We recently identified the p16INK4A tumor suppressor gene as one of the major target genes in this model, and showed by the use of in situ hybridization that allelic loss of p16IK4A occurs in the increased fraction of renal tubular cells within a few weeks. In the present study, we tested whether diets including green tea powder or a processed grain food are effective chemopreventive agents in this animal model. Consumption of these modified diets led to a significant decrease in the fraction of aneuploid cells after 1 week of repeated Fe-NTA administration. A decrease in renal lipid peroxidation after a single administration of Fe-NTA was also observed. Therefore, intake of green tea or processed grain foods stabilizes p16INK4A in the genome, at least in this model, and might be helpful for the prevention of ROS-associated cancer. This novel method is versatile, and may work as a surrogate end-point biomarker for screening the usefulness of agents for cancer chemoprevention.

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.

The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle.

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.

DNA recognition by the human UV-damaged DNA-binding (UV-DDB) protein was characterized. By circular permutation analyses, DNA duplexes containing the (6-4) photoproduct and the abasic site analog were found to be bent at angles of 54 degrees and 57 degrees, respectively, when they formed a complex with this protein. Although kinked NMR structures have been reported, fluorescence resonance energy transfer experiments revealed that these duplexes had no intrinsic bend. These results suggest that the UV-DDB protein binds DNA that can be bent easily at the above angle.

In the solid-phase synthesis of oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct using a dinucleotide building block, considerable amounts of by-products were found as the chain length increased. The by-products were the major product when a 49mer was synthesized on a 40 nmol scale. It was assumed that these by-products were formed by the coupling of phosphoramidites with the N3 imino function of the 5' component of the (6-4) photoproduct. We examined imidazolium triflate and benzimidazolium triflate to find an alternative activator for DNA synthesis. Imidazolium triflate prevented by-product formation to some extent, but the coupling yields were low. Benzimidazolium triflate was comparable to tetrazole in coupling efficiency and reduced by-product formation to a great extent, without modification of the synthesizer program. The obtained 49mer was used to detect proteins that recognize UV-damaged DNA in HeLa cell extracts. Two major protein-DNA complexes were found when a 49mer duplex was used as probe, while a 30mer duplex failed to detect one of them. This application showed the usefulness of long chain 'damaged' oligonucleotides in biochemical studies.

Distamycin A binds to DNA containing the (6-4) photoproduct, a major UV lesion that is recognized by the damaged DNA-binding (DDB) protein in human cells. We analyzed the binding properties of distamycin A and compared the results with those of the DDB protein. Structural change of the DNA duplex was not observed for distamycin A in two types of experiments, whereas the protein induced a large bending of the helix. Although the substrate specificity was different between the drug and the protein, thymine glycol was recognized by both of them, and inhibition of the DDB protein binding to the (6-4) photoproduct-containing DNA by distamycin A was tested.

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 → Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 → Asn substitution as well as Ser195 → Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH 2 ) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

In order to gain insight into the mechanism of mutations induced by photolesions in DNA, the thermal melting curves of oligonucleotide duplexes containing the (6-4) photoproducts of TT and TC and the Dewar isomer were measured at varying concentrations, and the thermodynamic parameters were determined. The duplexes were designed to be used as models for template-primer systems. In the case of d(CAXYAGCACGAC) x d(GTCGTGCTN), in which XY = T(6-4)T, T(6-4)C, or T(Dewar)T and N = A, G, C, or T, the -deltaG degrees values were the largest when N = G, and the difference between N = G and N = C or T was 1.1, 0.8, and 0.5 kcal/mol at 25 degrees C for XY = T(6-4)T, T(6-4)C, and T(Dewar)T, respectively. These results provide evidence of base pair formation between the 3'-pyrimidone and the opposite guanine, which was proposed previously to explain the T --> C transition induced at the 3' side of the T(6-4)T photoproduct. This interaction was weakened by isomerization to the Dewar photoproduct, which would explain the lower mutation specificity. For d(CAXYAGCACGAC) x d(GTCGTGCTGN), no obvious differences were observed regarding the base opposite the 5'-pyrimidine of the photoproducts. In contrast to the tertiary structure, determined by NMR, of a duplex containing T(6-4)T in its center [Kim, J.-K., & Choi, B.-S. (1995) Eur. J. Biochem. 228, 849-854], base pair formation at the 5'-pyrimidine was not detected for any photoproduct used in this study, even when the base opposite the 3'-pyrimidone of T(6-4)T was changed to adenine.