WEKO3
アイテム
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This covalentry linked dimer influences the replication and transcription, and leads\u003cbr /\u003eto cell death or skin cancer. Most (70-80%) of UV-induced DNA lesions is cyclobutane\u003cbr /\u003epyrimidine dimer (CPD) and, a lesser extent (20-30%) is (6-4) photoproduct. These two\u003cbr /\u003emajor types of UV-damaged DNA are repaired under illumination with near-UV/visible\u003cbr /\u003elight by CPD photolyase and (6-4) photolyase, respectively. The both photolyases contain\u003cbr /\u003eflavin adenine dinucleotide (FAD) as an essential cofactor for DNA repairing. The structure\u003cbr /\u003eand catalytic mechanism of CPD photolyase have been extensively studied. However, its\u003cbr /\u003edetailed structure of the active site containing CPD is uncertain until now. The structure of \u003cbr /\u003e(6-4) photolyase is less well studied. The genes for this enzyme exhibit a sequence\u003cbr /\u003esimilarity to CPD photolyase, especially in the FAD binding sites. Such high similarity\u003cbr /\u003eindicates a similar structure and reaction mechanism in two photolyases. Unexpectedly,\u003cbr /\u003e(6-4) photolyase presents a much lower quantum yield compared to that of CPD photolyase,\u003cbr /\u003eindicating the differences in structure and reaction mechanism between (6-4) and CPD\u003cbr /\u003ephotolyase. Such precise differences between CPD photolyase and (6-4) photolyase\u003cbr /\u003eregarding substrate binding and DNA repair needs to be clarified.\u003cbr /\u003e To investigate the unclear structure and environment of the active site in (6-4)\u003cbr /\u003ephotolyase, we measured resonance Raman spectra of (6-4) photolyase having neutral\u003cbr /\u003esemiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by\u003cbr /\u003eexcitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the\u003cbr /\u003efirst time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606\u003cbr /\u003ecm\u003csup\u003e-1\u003c/sup\u003e in H\u003csmall\u003e2\u003c/small\u003eO. whose frequency is the lowest among various flavoenzymes, apparently splits\u003cbr /\u003einto two comparable bands at 1594 and 1608 cm\u003csup\u003e-1\u003c/sup\u003e in D\u003csmall\u003e2\u003c/small\u003eO, and similarly that at 1522 cm\u003csup\u003e-1\u003c/sup\u003e in \u003cbr /\u003eH\u003csmall\u003e2\u003c/small\u003eO does into three bands at 1458, 1508, and 1536 cm\u003csup\u003e-1\u003c/sup\u003e in D\u003csmall\u003e2\u003c/small\u003eO. This D\u003csmall\u003e2\u003c/small\u003eO effect was \u003cbr /\u003erecognized only after being oxidized once and photoreduced to form a semiquinone again,\u003cbr /\u003ebut not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were\u003cbr /\u003eobserved at significantly low frequencies (1621, 1576 cm\u003csup\u003e-1\u003c/sup\u003e)and with band splittings\u003cbr /\u003e(1508/1493, 1346/1320 cm\u003csup\u003e-1\u003c/sup\u003e). These Raman spectral characteristics indicate strong H-bonding\u003cbr /\u003einteractions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in\u003cbr /\u003ebenezene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.\u003cbr /\u003e To clarify the structure of active site upon substrate binding and the mechanism of DNA repair, we\u003cbr /\u003eexamined the resonance Raman spectra of complexes between damaged DNA and the neutral\u003cbr /\u003esemiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral\u003cbr /\u003esemiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the\u003cbr /\u003ebenzene ring of flavin adenine dinucleotide (FAD), were shifted to lower freauencies upon binding of \u003cbr /\u003edamaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the\u003cbr /\u003ebenzene ring of FAD directly but that (6-4) photoproduct does not. Bands II and VII of the oxidized\u003cbr /\u003eflavin and the 1398/1391 cm\u003csup\u003e-1\u003c/sup\u003e bands of the neutral semiquinoid flavin, which may reflect the bending of\u003cbr /\u003ethe U-shaped FAD, were altered upon substrate binding suggesting that CPD and (6-4) photoproduct\u003cbr /\u003einteract with the adenine ring of FAD, When substrate is bound, there is an upshiftesd 1528 cm\u003csup\u003e-1\u003c/sup\u003e band of\u003cbr /\u003ethe neutral semiquinoid flavin in CPD photolyase, indicating a weekend hydrogen bond at N5-H of\u003cbr /\u003eFAD, and in (6-4) photolyase, band X is downshifted, indicating a strengthened hydrogen bond at N3-H\u003cbr /\u003eof FAD. These Raman spectra led us to conclude that the two photolyases have different electron \u003cbr /\u003etransfer mechanisms as well as different hydrogen bonding environments, which account for the higher\u003cbr /\u003eredox potential of CPD photolyase.\u003cbr /\u003e This work revealed that the FAD in (6-4) photolyase is characterized by an electron\u003cbr /\u003elocalized structure, and binds to the protein in a fairly hydrophobic and strong hydrogen \u003cbr /\u003ebonding environment, Specially, a stronger H-bonding at N5-H of FAD was identified for \u003cbr /\u003e(6-4) photolyase, which may result in the low quantum yield for DNA-repair of this\u003cbr /\u003eenzyme. Besides, UV-damaged DNA contacts the benzene ring of FAD only in CPD\u003cbr /\u003ephotolyase and the adenine ring of FAD in both photolyases. These structures indicate that\u003cbr /\u003e the election transfer during DNA -repair between isoalloxazine and UV-damaged DNA in \u003cbr /\u003eCPD photolyase is direct, whereas that in (6-4) photolyase is not direct and bridged by\u003cbr /\u003eadenine.", "subitem_description_type": "Other"}]}, "item_1_description_18": {"attribute_name": "フォーマット", "attribute_value_mlt": [{"subitem_description": "application/pdf", "subitem_description_type": "Other"}]}, "item_1_description_7": {"attribute_name": "学位記番号", "attribute_value_mlt": [{"subitem_description": "総研大甲第1014号", "subitem_description_type": "Other"}]}, "item_1_select_14": {"attribute_name": "所蔵", "attribute_value_mlt": [{"subitem_select_item": "有"}]}, "item_1_select_8": {"attribute_name": "研究科", "attribute_value_mlt": [{"subitem_select_item": "先導科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "22 光科学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "2006"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "LI, Jiang", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "0", "nameIdentifierScheme": "WEKO"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 0, "filename": "甲1014_要旨.pdf", "filesize": [{"value": "312.3 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 312300.0, "url": {"label": "要旨・審査要旨", "url": "https://ir.soken.ac.jp/record/1250/files/甲1014_要旨.pdf"}, "version_id": "a4884a63-7dd4-4707-9690-2d6fd958b946"}, {"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2016-02-17"}], "displaytype": "simple", "download_preview_message": "", "file_order": 1, "filename": "甲1014_本文.pdf", "filesize": [{"value": "11.1 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 11100000.0, "url": {"label": "本文", "url": "https://ir.soken.ac.jp/record/1250/files/甲1014_本文.pdf"}, "version_id": "2d1248dc-9951-4550-87c4-eca306756954"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "eng"}]}, "item_resource_type": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"resourcetype": "thesis", "resourceuri": "http://purl.org/coar/resource_type/c_46ec"}]}, "item_title": "Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA"}, {"subitem_title": "Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "1", "path": ["24"], "permalink_uri": "https://ir.soken.ac.jp/records/1250", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "1250", "relation": {}, "relation_version_is_last": true, "title": ["Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA"], "weko_shared_id": -1}
Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA
https://ir.soken.ac.jp/records/1250
https://ir.soken.ac.jp/records/1250b3fcf8dd-0aa0-4c94-8b2d-f1b3e671d04c
名前 / ファイル | ライセンス | アクション |
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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公開日 | 2010-02-22 | |||||
タイトル | ||||||
タイトル | Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | Resonance Raman Studies on Reaction Mechanism of Photolyases: Structural Characteristics of the Active Site and Photo-repair Mechanism of UV-damaged DNA | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
資源タイプ | thesis | |||||
著者名 |
李, 江
× 李, 江 |
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フリガナ |
リ, ジャン
× リ, ジャン |
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著者 |
LI, Jiang
× LI, Jiang |
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学位授与機関 | ||||||
学位授与機関名 | 総合研究大学院大学 | |||||
学位名 | ||||||
学位名 | 博士(理学) | |||||
学位記番号 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 総研大甲第1014号 | |||||
研究科 | ||||||
値 | 先導科学研究科 | |||||
専攻 | ||||||
値 | 22 光科学専攻 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 2006-09-29 | |||||
学位授与年度 | ||||||
2006 | ||||||
要旨 | ||||||
内容記述タイプ | Other | |||||
内容記述 | Ultraviolet light radiation in the wavelength range between 260 and 320 nm causes<br />damage to DNA by forming the dimerization of adjacent pyrimidines in the same DNA<br />strand. This covalentry linked dimer influences the replication and transcription, and leads<br />to cell death or skin cancer. Most (70-80%) of UV-induced DNA lesions is cyclobutane<br />pyrimidine dimer (CPD) and, a lesser extent (20-30%) is (6-4) photoproduct. These two<br />major types of UV-damaged DNA are repaired under illumination with near-UV/visible<br />light by CPD photolyase and (6-4) photolyase, respectively. The both photolyases contain<br />flavin adenine dinucleotide (FAD) as an essential cofactor for DNA repairing. The structure<br />and catalytic mechanism of CPD photolyase have been extensively studied. However, its<br />detailed structure of the active site containing CPD is uncertain until now. The structure of <br />(6-4) photolyase is less well studied. The genes for this enzyme exhibit a sequence<br />similarity to CPD photolyase, especially in the FAD binding sites. Such high similarity<br />indicates a similar structure and reaction mechanism in two photolyases. Unexpectedly,<br />(6-4) photolyase presents a much lower quantum yield compared to that of CPD photolyase,<br />indicating the differences in structure and reaction mechanism between (6-4) and CPD<br />photolyase. Such precise differences between CPD photolyase and (6-4) photolyase<br />regarding substrate binding and DNA repair needs to be clarified.<br /> To investigate the unclear structure and environment of the active site in (6-4)<br />photolyase, we measured resonance Raman spectra of (6-4) photolyase having neutral<br />semiquinoid and oxidized forms of FAD, which were selectively intensity enhanced by<br />excitations at 568.2 and 488.0 nm, respectively. DFT calculations were carried out for the<br />first time on the neutral semiquinone. The marker band of a neutral semiquinone at 1606<br />cm<sup>-1</sup> in H<small>2</small>O. whose frequency is the lowest among various flavoenzymes, apparently splits<br />into two comparable bands at 1594 and 1608 cm<sup>-1</sup> in D<small>2</small>O, and similarly that at 1522 cm<sup>-1</sup> in <br />H<small>2</small>O does into three bands at 1458, 1508, and 1536 cm<sup>-1</sup> in D<small>2</small>O. This D<small>2</small>O effect was <br />recognized only after being oxidized once and photoreduced to form a semiquinone again,<br />but not by simple H/D exchange of solvent. Some Raman bands of the oxidized form were<br />observed at significantly low frequencies (1621, 1576 cm<sup>-1</sup>)and with band splittings<br />(1508/1493, 1346/1320 cm<sup>-1</sup>). These Raman spectral characteristics indicate strong H-bonding<br />interactions (at N5-H, N1), a fairly hydrophobic environment, and an electron-lacking feature in<br />benezene ring of the FAD cofactor, which seems to specifically control the reactivity of (6-4) photolyase.<br /> To clarify the structure of active site upon substrate binding and the mechanism of DNA repair, we<br />examined the resonance Raman spectra of complexes between damaged DNA and the neutral<br />semiquinoid and oxidized forms of (6-4) and CPD photolyases. The marker band for a neutral<br />semiquinoid flavin and band I of the oxidized flavin, which are derived from the vibrations of the<br />benzene ring of flavin adenine dinucleotide (FAD), were shifted to lower freauencies upon binding of <br />damaged DNA by CPD photolyase but not by (6-4) photolyase, indicating that CPD interacts with the<br />benzene ring of FAD directly but that (6-4) photoproduct does not. Bands II and VII of the oxidized<br />flavin and the 1398/1391 cm<sup>-1</sup> bands of the neutral semiquinoid flavin, which may reflect the bending of<br />the U-shaped FAD, were altered upon substrate binding suggesting that CPD and (6-4) photoproduct<br />interact with the adenine ring of FAD, When substrate is bound, there is an upshiftesd 1528 cm<sup>-1</sup> band of<br />the neutral semiquinoid flavin in CPD photolyase, indicating a weekend hydrogen bond at N5-H of<br />FAD, and in (6-4) photolyase, band X is downshifted, indicating a strengthened hydrogen bond at N3-H<br />of FAD. These Raman spectra led us to conclude that the two photolyases have different electron <br />transfer mechanisms as well as different hydrogen bonding environments, which account for the higher<br />redox potential of CPD photolyase.<br /> This work revealed that the FAD in (6-4) photolyase is characterized by an electron<br />localized structure, and binds to the protein in a fairly hydrophobic and strong hydrogen <br />bonding environment, Specially, a stronger H-bonding at N5-H of FAD was identified for <br />(6-4) photolyase, which may result in the low quantum yield for DNA-repair of this<br />enzyme. Besides, UV-damaged DNA contacts the benzene ring of FAD only in CPD<br />photolyase and the adenine ring of FAD in both photolyases. These structures indicate that<br /> the election transfer during DNA -repair between isoalloxazine and UV-damaged DNA in <br />CPD photolyase is direct, whereas that in (6-4) photolyase is not direct and bridged by<br />adenine. | |||||
所蔵 | ||||||
値 | 有 | |||||
フォーマット | ||||||
内容記述タイプ | Other | |||||
内容記述 | application/pdf |