京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19068号 / 人博第721号 / 新制||人||173(附属図書館) / 26||人博||721(吉田南総合図書館) / 32019 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)教授 小松 賢志, 教授 宮下 英明, 准教授 三浦 智行 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.

Homologous recombination (HR) is essential for the repair of DNA doublestrand breaks (DSBs) and damaged replication forks. However, HR can also cause gross chromosomal rearrangements (GCRs) by producing crossovers (COs), resulting in the reciprocal exchange of sequences between non-sister chromatids. Therefore, HR-mediated GCRs are suppressed via the promotion of HR pathways that favour noncrossover (NCO) formation, such as the synthesis-dependent strand annealing (SDSA) and dissolution pathways, which are modulated by Mph1 and Sgs1 helicases, respectively. The mismatch repair (MMR) pathway is intricately associated with HR via its roles in repairing mismatches on heteroduplex DNA that can arise during HR and in preventing homeologous recombination. Using a plasmid break-repair assay, we have revealed a novel, MMR-independent role of MutSα in promoting the formation of a subset of COs that is specifically supressible by Mph1, during HR between two completely homologous sequences. In contrast, the MMR-dependent function of MutSα, together with Mph1 and Sgs1, was shown to be required for the suppression of CO formation during homeologous recombination. These data indicate that Mph1 can both antagonise and promote the functions of MutSα during DSB repair, depending on the levels of homology between the two recombining sequences. COs are generated by the resolution of Holliday junction (HJ) intermediates formed at the terminal stages of HR. Several S.cerevisiae proteins such as Yen1, Mus81, Slx1 and Rad1 have been implicated in HJ resolution. However, the in vivo roles of these proteins in HJ resolution remain to be confirmed. To directly and quantitatively monitor in vivo HJ resolution in S.cerevisiae, a transformation-based HJ resolution assay using a plasmid-borne HJ substrate has been developed. Using this system, we have demonstrated an in vivo HJ resolution function of Yen1, which acts redundantly with Mus81. Moreover, these redundant activities of Yen1 and Mus81 are essential for survival during replication stress, but are dispensable for DSB repair. An Slx4 and Rad1-dependent in vivo HJ resolution activity was also observed in the absence of Yen1 and Mus81 that was suppressed by presence of Slx1. Models describing how the nucleases interact to process HJs in vivo will be discussed.

Due to the action of endogenous and exogenous agents, DNA is subject up to 70,000 lesions per day, thus the existence of repair mechanisms and enzymes is more than necessary. We know basic mechanisms of several specific DNA repair pathways, of which the Fanconi anaemia (FA) repair pathway is one of the least explored. FA is a rare, autosomal recessive disorder characterized by early onset bone marrow failure, developmental defects, genomic instability and predisposition to acute myeloid leukaemia and solid tumours. The primary diagnosis of FA is a hypersensitivity to cross-linking agents of DNA due to inactivation of one of the 21 genes from the FA repair pathway, the so-called FANC genes (FA complementation group). The molecular defect in FA is an impaired repair of DNA interstrand cross-links (ICLs). The ICLs are cytotoxic lesions that inhibit the process of DNA replication and transcription. A crucial step in the FA pathway that initiates ICL repair is a monoubiquitination of FANCD2. FANCD2 monoubiquitination is a base for the recruitment of additional proteins that coordinate DNA repair. Ubiquitin is recruited via activating enzyme E1 (UBA1), ubiquitin-conjugating enzyme E2T (UBE2T) and transferred onto FANCD2 by multisubunit E3 ligase (FA core complex). There are up to 11 different proteins...

Fanconi anemia is an autosomal recessive disorder caused by mutation in one of Fanconi genes and it is manifested by developmental abnormalities, bone marrow failure, predisposition to cancer, cellular sensitivity to cross-linking agents and many other symptoms. Proteins encoded by Fanconi genes and some other proteins are part of Fanconi anemia pathway (FA pathway), which is responsible for DNA repair of an interstrand cross-link (ICL). The repair by this pathway requires monoubiquitination of FANCD2, which is induced and regulated by ATR dependent FANCI phosphorylation. The FANCI phosphorylation initiates the FA pathway but the molecular mechanism of this initialization is not known. Furthermore the proper function of entire pathway requires both: sequence of phosphorylation events of FANCI and monoubiquitination of FANCI:FANCD2 complex . The principle of this work was to study molecular mechanism of initiation and regulation of FA pathway by FANCI phosphorylation. Therefore phosphomimetic mutants of FANCI have been created to investigate their role in processes leading to FANCD2 monoubiquitination. The main aim was to reveal how the phosphorylation of FANCI affects DNA binding and also DNA binding of FANCI:FANCD2 complex. Since both DNA and FANCI phosphorylation are required for proper FANCD2...

L'objectiu d'aquest tesi és avaluar els diferents passos en el camí que va des de l'exposició a contaminants atmosfèrics/PAHs fins a la malaltia, el càncer de bufeta urinària. Es van mesurar partícules ultrafines a Barcelona. S'ha avaluat l'exposició a contaminació atmosfèrica en un estudi cas-control, recollint informació sobre la història residencial incloent diversos indicadors de l'exposició a contaminació atmosfèrica i altres factors de risc potencials. Es va dur a terme una revisió sistemàtica de la literatura per avaluar si els nivells de metabòlits del pirè i els aductes d'ADN i de proteïnes es correlacionaven amb nivells baixos d'exposició a PAHs. Vam mesurar els nivells d'aductes d'ADN en un subgrup d'individus de l'estudi cas-control amb la tècnica del radioetiquetatge amb fòsfor-32, tractament de la nucleasa P1. Vam analitzar 22 SNPs en set gens de la via de reparació de l'ADN per excisió de nucleòtids. / The aim of this thesis is to evaluate the different steps in the pathway from exposure (air-contaminants/PAHs) to disease (bladder cancer). We measured ultrafine particles in Barcelona. We evaluated the exposure to air pollutants in a case-control study, collecting information on the residential history with proxies for exposure to air pollution and other potential risk factors. We did a systematic review of the literature to evaluate if pyrene metabolites and DNA and protein adducts are correlated with low level exposure to PAHs. We measured bulky DNA adducts in a subgroup of subjects of the case-control study using 32P-Postlabeling, nuclease P1 treatment. We analyzed 22 SNPs in 7 genes of the nucleotide excision repair pathway.

Thyroid hormones, which are secreted by the thyroid gland, are one of the most important halogenated compounds in the body. Thyroid hormones control almost every processes in the body including growth, body temperature, protein synthesis, carbohydrate and fat metabolism, heart rate, and cardiovascular, renal and brain function. Thyroid gland secretes L-thyroxine or 3,3',5,5'-tetraiodothyronine (T4) as a prohormone. While the biologically active hormone 3,3',5-triiodothyronine (T3) is produced by selective phenolic ring deiodination of T4, selective tyrosyl ring deiodination of T4 produces a biologically less active metabolite 3,3',5'-triiodothyronine (rT3). Tyrosyl and phenolic ring deiodination of T3 and rT3, respectively, also produces a biologically inactive metabolite 3,3'-diiodothyronine (3,3'-T2). Regioselective deiodinations of thyroid hormones are catalysed by three isoforms of a selenoenzyme iodothyronine deiodinase (DIO1, DIO2, DIO3). DIO1 can remove iodine from both the tyrosyl and phenolic rings of thyroid hormones, whereas DIO2 and DIO3 are selective towards phenolic and tyrosyl ring, respectively. Although the Figure 1. (A) Deiodination of thyroid hormones by iodothyronine deiodinases (DIOs) (A) and naphthyl-based selenium and/or sulphur compounds (B). mystery behind the origin of regioselectivity of deiodination by DIOs remains unsolved, formation of halogen bonding between selenium in the active site of DIOs and iodine of thyroid hormones has been widely accepted as the mechanism of deiodination. Halogen bonding, a noncovalent interaction between halogen and an electron donor such as nitrogen, oxygen, sulphur, selenium etc., elongates the C-I bond and impart a carbanionic character on the carbon atom that gets protonated after the removal of iodide. Apart from the deiodination, thyroid hormones also undergo decarboxylation, oxidative deamination, sulphate-conjugation to form iodothyronamines, iodothyroaetic acids and sulphated thyroid hormones, respectively. Figure 2. (A) Proposed mechanism of deiodination of thyroid hormones by deiodinase mimics. (B) Halogenation of uracil- and cytosine-containing nucleosides by hypohalous acid (HOX). Recently, naphthyl-based selenium/sulphur-containing compounds, such as compound 1 (Figure 1B), have been reported to mediate the selective tyrosyl ring deiodination of T4 and T3 to form rT3 and 3,3'-T2, respectively. Interestingly, replacement of the selenol moiety in compound 1 with a thiol decreases the activity, whereas replacement of the thiol moiety with another selenol dramatically increases the deiodination activity. Based on the detailed experimental and theoretical investigations, a mechanism involving the Se···I halogen bonding was proposed (Figure 2A). In addition to the halogen bonding between selenium and iodine atom, chalcogen bonding between two nearby chalcogen atoms was also shown to be important for the deiodination activity. Another important class of halogenated compounds in the body are the halogenated nucleosides. Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes, which can convert halide ions (X¯) into a toxic reactive halogen species hypohalous acid (HOX) in presence of hydrogen peroxide (H2O2). Uracil- and cytosine-containing nucleosides are known to undergo halogenation at the 5-position of the nucleobase to form the halogenated nucleosides (Figure 2B). Interestingly, halogenated nucleosides such as 5-halo-2'-deoxyuridine are known to be incorporated in the DNA of dividing cells essentially substituting for thymidine. Incorporation of halogenated nucleosides into the DNA leads to mutagenesis, carcinogenesis and loss of genome integrity. Thymidylate synthase (TSase), the key enzyme involved in the biosynthesis of 2'-deoxythmidine-5'-monophosphate (dTMP) from 2'-deoxyuridine-5'-monophosphate (dUMP), can catalyse the dehalogenation of halogenated nucleotides in presence of external thiols. This thesis consists of five chapters. The first chapter provides a general introduction to halogen bonding, thyroid hormones and halogenated nucleosides. This chapter also briefly describes the halogen bond-mediated biochemical and biomimetic deiodinations of thyroid hormones by iodothyronine deiodinases and naphthyl-based organoselenium compounds. Dehalogenation of halogenated nucleotides by thymidylate synthase and thiol-based small molecules has also been discussed in this chapter. The second chapter of this thesis contains the regioselective deiodination of iodothyronamines (TAMs) by deiodinases mimics. TAMs are the endogenous metabolites produced by the decarboxylation of β-alanine side chain of thyroid hormones (THs). 3,3',5-triiodothyronamine (T3AM) and 3,5-diiodothyronamine (3,5-T2AM) undergoes selective tyrosyl ring deiodination by deiodinase mimics to form 3,3'-diiodothyronamine (3,3'-T2AM) and 3-iodothyronamine (3-T1AM), respectively. Interestingly, when the initial rates of deiodinations of T3 and T3AM were compared, deiodination of T3 was found to be several fold faster than that of T3AM under identical reaction conditions. To understand the ability of the iodine atoms to form Figure 3. (A) HPLC chromatogram of deiodination of T3. (B) Proposed mode of interaction of dimeric T3 and monomeric T3AM with organoselenium compounds. halogen bonding, a model selenolate (MeSe¯) was optimized with the T3 and T3AM. Although both T3 and T3AM forms the expected Se···I halogen bonding with MeSe¯, the strength of halogen bonding was found to be less for T3AM than T3. Furthermore, detailed kinetic and spectroscopic studies indicate that T3 and T3AM exist as dimeric and monomeric species in solution. The dimerization of T3 in solution was shown to have remarkable impact on the activation energy and pre-exponential factor of the deiodination reactions. Single crystal X-Ray crystallography and theoretical calculations indicated that in addition to Se···I halogen bonding, I···I halogen bonding may play an important role in the deodination of thyroid hormones by deiodinase mimics. Furthermore, the presence of heteroatoms such as nitrogen, oxygen and sulphur in the close proximity of one of the selenium atoms of deiodinase mimics was shown to have significant effect on the rate of deiodination reactions. The third chapter of the thesis focusses on the conformational polymorphism and conformation-dependent halogen bonding of L-thyroxine. Synthetic version of L-thyroxine (T4) is a life-saver for millions of people who are suffering from hypothyroidism, a thyroidal disorder recognised by low levels of T4 and elevated levels of TSH in blood plasma. Synthetic version of L-thyroxine is available in the Figure 4. Ball and stick model of the single crystal X-Ray structure of the conformational polymorphs of L-thyroxine. Form I and Form II was exclusively crystallized from methanol and acetonitrile, respectively. Water molecules are omitted for clarity. market with various brand names. However, adverse effects have been observed in the patients when they switch their brand of thyroxine. Based on these observations, the American Thyroid Association (ATA), the Endocrine Society (TES), and the American Association of Clinical Endocrinologists (AACE) declared that the different brands of T4 are not bioequivalent, thus leading to differences in the bioavailability of the drug. We have shown that the commercially available thyroxine exists in at least two stable forms (Form I and Form II) with different three-dimensional structures (Figure 4). These two forms exhibit different intermolecular interactions in crystal packing, spectral behaviours, thermal stabilities, optical activity and very interestingly, different solubility in acidic and basic pH. At pH 4, solubility of Form I is about 42% and 45% greater than that of Form II and bulk T4, respectively, whereas at pH 9, the solubility of Form II is about 38% and 42% higher than that of Form I and bulk T4, respectively. As T4 is a narrow therapeutic index drug, these differences in solubility may have remarkable impact on the bioavailability of the drug. In addition to this, we have shown that the ability of the iodine atoms in the C-I bonds to form halogen bond with donor atoms can be altered by changing the relative orientation of tyrosyl and phenolic rings in T4. In the fourth chapter, the three-dimensional structures and conformations of thyroid hormones (THs) and iodothyronamines (TAMs) are discussed. TAMs, the endogenous decarboxylated metabolites of THs, exhibit different binding affinities to the transport proteins and iodothyronine deiodinases (DIOs) compared to the THs. Figure 5. Change in the structure and conformations of thyroid hormones and iodothyronamines with the decarboxylation of amino acid side chain and deiodination of phenolic and tyrosyl ring. Furthermore, the substrate specificities of DIOs have been found to be dependent on the position of iodine atoms on the phenolic and tyrosyl ring of TAMs and THs. Single crystal X-ray structures of TAMs indicate that decarboxylation of amino acid side chain of THs induces significant changes in the structure and conformation. Furthermore, the positional isomers of THs and TAMs exhibit remarkably different conformations, which may have significant effect on the binding of these metabolites to the active site of DIOs. In addition to the structure and conformations, different categories of the intermolecular halogen···halogen (X···X) interactions in the crystal packing of THs and TAMs have also been discussed. Natural bond orbital (NBO) analysis have been done on the halogen-bonded geometries to understand the electronic nature of these interactions. In the fifth chapter, the dehalogenation of halogenated nucleosides and nucleobases by naphthyl-based sulphur/selenium compounds is discussed. Purine and pyrimidine nucleosides are halogenated at various positions of the aromatic ring by different peroxidases such as myeloperoxidase and eosinophil peroxidase present in the white blood cells. Incorporation of the halogenated nucleosides into the DNA of replicating cells leads to DNA-strand breaks, mutagenesis, carcinogenesis and loss of Figure 6. (A) Dehalogenation of halogenated nucleosides. Effect of base-pairing wih adenine and guanine on the deiodination of IU (B) and debromination of BrU (C) by compound 2. genome integrity. We have shown that the naphthalene-based organoselenium compounds such as compound 2 can mediate the dehalogenation of 5-iodo-2'-deoxyuridine (5-IdUd) and 5-bromo-2'-deoxyuridine (5-BrdUd) to produce 2'-deoxyuridine (dUd) (Figure 6A). The deiodination of 5-IdUd was found to be faster than the debromination of 5-BrdUd by compound 2. The mechanism of dehalogenation of halogenated nucleosides by compound 2 was found to be dependent on the nature of halogen. While the deiodination of 5-IdUd by compound 2 follow halogen bond-mediated pathway like thyroid hormones, debromination of 5-BrdUd follow a Michael addition-elimination pathway. Similar results were obtained when 5-iodo-2'-deoxycytidine (5-IdCd) or 5-bromo-2'-deoxycytidine (5-BrdCd) was used as substrate for dehalogenation reaction. Base-pairing of 5-iodouracil (IU) and 5-bromouracil (5-BrU) with adenine and guanine has a significant effect on the rate of dehalogenations of IU and BrU by compound 2 (Figure 6B and 6C).

The thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined. The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed. Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point.

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Indiana University-Purdue University Indianapolis (IUPUI) / Translation Initiation in protein synthesis is a crucial step controlling gene expression that enhanced by eukaryotic translation initiation factors (eIFs). eIF3a, the largest subunit of eIF3 complexes, has been shown to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of proteins important for repair of double strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a up-regulation sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) activity by suppressing expression level of NHEJ repair proteins such as DNA-PKcs and vice versa. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies including ionizing radiation.