{"created":"2023-06-20T13:21:08.642291+00:00","id":1250,"links":{},"metadata":{"_buckets":{"deposit":"afa60141-b9b8-4717-b6e3-8b4d7f200ed6"},"_deposit":{"created_by":1,"id":"1250","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1250"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001250","sets":["2:431:24"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"李, 江"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"リ, ジャン"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2006-09-29"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　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.","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","filename":"甲1014_要旨.pdf","filesize":[{"value":"312.3 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","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","filename":"甲1014_本文.pdf","filesize":[{"value":"11.1 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","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"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1250","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_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:06:32.356813+00:00"}