DNA複製ライセンス化因子Cdt1の機能ドメインに関する解析

(1)

DNA複製ライセンス化因子Cdt1の機能ドメインに関

する解析

著者

川端亮介

学位授与機関

Tohoku University

(2)

➠ୌ❮ さ᪠ ࠘⫴ᬊ࠙ 々㞟࠾ࡗ㛏ኬ࡝┷ᰶ⏍∸ࡡ DNA ࡡ々⿿ࢅୌᅂࡡ⣵⬂࿔᭿࡚ධ㛏࡞ῳࡖ࡙ḿ ☔࡞ୌᅂࡡࡲ㐑⾔ࡌࡾࡒࡴࠉ┷ᰶ⏍∸࡞ࡢኣᒪ࡞ࢂࡒࡾཚᐠ࡝โᚒᶭᵋ࠿ങ࠻ ࡼࡿ࡙࠷ࡾࠊࡐࡿࡼࡡโᚒᶭᵋࡡୌࡗ࡛ࡊ࡙DNA 々⿿ࣚ࢕ࢬࣤࢪ໩ᶭᵋ࠿Ꮛᅹ ࡊࠉDNA 々⿿ࡡ㛜ጙࡡโᚒࡷ්々⿿ࡡᢒḾ࡞㔔さ࡝ᙲ๪ࢅᯕࡒࡊ࡙࠷ࡾࡆ࡛࠿ ▩ࡼࡿ࡙࠷ࡾࠊࣚ࢕ࢬࣤࢪ໩ཬᚺࡢ⣵⬂࿔᭿ࡡิ᭿࡞࠽ࡄࡾ々⿿㉫Ⅴ࡫ࡡ々⿿ ㉫Ⅴヾㆉ々ྙమ (ORC) ࡡ⤎ྙ࡞ጙࡱࡽࠉORC ࡡ⤎ྙ࡞౪Ꮛࡊ࡙ Cdc6 ࡛ Cdt1 ࠿ࢠ࣏ࣞࢲࣤ୕࡫⤎ྙࡊࡒࡡࡔࠉࡆࡿࡼࡡࢰࣤࣂࢠ㈻ࡡᶭ⬗࡞ࡻࡖ࡙ Mcm2-7 々ྙమ࠿ࢠ࣏ࣞࢲࣤ୕࡞ᑙථࡈࡿࡾࡆ࡛࡞ࡻࡽᏰ஡ࡌࡾࠊ ࡆࡿࡱ࡚࡞ᙔ◂✪ᐄ࡞ࡻࡽࠉCdc6 ࡛ Cdt1 ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡵ㡨ᗆ࠿Ꮛᅹ ࡊࠉࡆࡡ㡨ᗆ࠿஗ࡿࡒ㝷࡞ࡢࠉࡆࡿࡼࡡࢰࣤࣂࢠ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡢ≁࡞ ᙫ㡢࠿࡝࠷࡞ࡵ㛭ࢂࡼࡍࠉࣚ࢕ࢬࣤࢪ໩࠿Ᏸ஡ࡊ࡝࠷ࡆ࡛ࢅሒ࿈ࡊ࡙࠷ࡾࠊࡌ ࡝ࢂࡔࠉCdt1 ࡛ Cdc6 ࡢరࡼ࠾ࡡ┞பష⏕ࢅ᭯ࡊࠉࣚ࢕ࢬࣤࢪ໩࡞࠽࠷࡙༝ㄢ Ⓩ࡞ᶭ⬗ࡊ࡙࠷ࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊࡐࡆ࡚ᮇ◂✪࡚ࡢࠉCdt1 ࡛ Cdc6 ࡡࢰ ࣤࣂࢠ㈻㛣┞பష⏕ࡡレ⣵࡝よᯊࢅ㏳ࡋ࡙ࠉࣚ࢕ࢬࣤࢪ໩ࡡฦᏄᶭᗆࢅ⌦よࡌ ࡾࡆ࡛ࢅ┘Ⓩ࡛ࡊࠉ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ㸝Xenopus㸞༵᢫ฝᾦ↋⣵⬂ᐁ㥺⣌ࢅ ⏕࠷ࡒ⏍໩ᏕⓏよᯊࢅ⾔ࡖࡒࠊ ࠘⤎ᯕ࣬⩻ᐳ࠙ 1ࠉCdt1-Cdc6 ࢰࣤࣂࢠ㈻㛣┞பష⏕ࡡよᯊ Cdt1 ࢰࣤࣂࢠ㈻ࢅ N ᮆ❻ഁ (Cdt1-N; 1㹳243 a.a)ࠉ୯ኳ (Cdt1-M; 243㹳447

a.a)ࠉC ᮆ❻ഁ (Cdt1-C; 447㹳621 a.a) ࡡ㸨ࡗࡡ㡷ᇡ࡞ฦࡄࠉHis-tag ⼝ྙᆵࢰ

(3)

࡛ࡡ┞பష⏕࠿ふᐳࡈࡿࡒࠊࡈࡼ࡞ୌᏽࡡ⃨ᗐࡡHis-Cdt1 ࡛ GST-Cdc6 Ꮛᅹୖ ࡞ࠉHis-Cdt1-M ࢅ῟ຊࡌࡾ࡛ࠉHis-Cdt1-M ࡡ⃨ᗐࡡ୕࡛࡛᪴ࡵ࡞ࠉGST-Cdc6 ࡫ࡡ His-Cdt1 ࡡ⤎ྙ㔖࠿఩ୖࡌࡾࡆ࡛࠿♟ࡈࡿࡒࠊ௧୕ࡻࡽࠉCdt1 ࡢࡐࡡ୯ ኳ㡷ᇡ࡚Cdc6 ࡛┞பష⏕ࡌࡾࡆ࡛࠿♟ြࡈࡿࡒࠊ 2ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡ DNA 々⿿࡫ࡡᙫ㡢 Cdt1 ࢅඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ࡞୕オࡡ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒ࡛ࡆ ࢀࠉMcm6 ࡡࢠ࣏ࣞࢲࣤ⤎ྙࡢ☔ヾࡈࡿࡍࠉDNA 々⿿Ὡᛮࡵヾࡴࡼࡿ࡝࠾ࡖࡒ ࡆ࡛࠾ࡼࠉ࠷ࡍࡿࡡCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡵࣚ࢕ࢬࣤࢪ໩Ὡᛮࢅᣚࡒ࡝࠷࡛⩻࠻ ࡼࡿࡒࠊࡐࡆ࡚ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ Cdt1 ࢅ㝎ཡࡊ࡙࠷࡝࠷༵᢫ฝᾦ࡞῟ ຊࡊࡒ࡛ࡆࢀࠉHis-Cdt1-NࠉHis-Cdt1-C ࡡ῟ຊ࡞ࡻࡾ DNA 々⿿Ὡᛮ࡫ࡡᙫ㡢 ࡢࡲࡼࡿ࡝࠾ࡖࡒ࠿ࠉHis-Cdt1-M ࡡ῟ຊ࡞ࡻࡖ࡙ DNA 々⿿࠿ᢒโࡈࡿࡒࠊ ࡗ࠷࡚ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒ༵᢫ฝᾦࢅ⏕࠷࡙ࠉࣚ࢕ࢬࣤࢪ໩࠿ Ᏸ஡ࡌࡾ20 ฦᚃ࡞࠽ࡄࡾ DNA 々⿿㛭㏻ࢰࣤࣂࢠ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡗ࠷࡙ ᳠ゞࡊࡒ࠿ࠉHis-Cdt1-M ῟ຊ࡞ࡻࡾ Mcm6 ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡫ࡡ㢟ⴥ࡝ᢒโ ࡢヾࡴࡼࡿ࡝࠾ࡖࡒࠊࡈࡼ࡞ࠉࣚ࢕ࢬࣤࢪ໩Ᏸ஡௧㜾࡞Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ ῟ຊ ࡊࡒ 㝷 ࡞ ࠽ࡄ ࡾ DNA 々⿿࡞࠵ࡒ࠻ࡾᙫ㡢ࢅ᳠ゞࡊࡒࠊࡐࡡ⤎ᯕࠉ His-Cdt1-M ࡢࣚ࢕ࢬࣤࢪ໩Ᏸ஡ᚃ࡚ࡵ DNA 々⿿Ὡᛮࢅ఩ୖࡈࡎࡾࡆ࡛࠿♟ြ ࡈࡿࡒࠊࡐࡆ࡚ࠉࣚ࢕ࢬࣤࢪ໩௧㜾ࡡDNA 々⿿ࡡṹ㝭࡞ᑊࡌࡾᙫ㡢࡞ࡗ࠷࡙ࡈ ࡼ࡞☔ヾࡊࡒ࡛ࡆࢀࠉDNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞ࡢᙫ㡢ࢅཀྵ࡯ࡈ࡝࠷࠿ࠉ Mcm2-7 々ྙమࡡὩᛮ໩ࡡᣞᵾ࡚࠵ࡾ Mcm4 ࡡࣛࣤ㓗໩࠿΅ᘽࡊ࡙࠷ࡾࡆ࡛ࠉ DNA polymerase α ࡡࢠ࣏ࣞࢲࣤ࠾ࡼࡡよ㞫࠿㐔ࡿ࡙࠷ࡾࡆ࡛࠿♟ြࡈࡿࡒࠊ ௧୕ࡻࡽࠉHis-Cdt1-M ࡞ࡻࡾ DNA 々⿿Ὡᛮ఩ୖࡡཋᅄࡢࠉࣚ࢕ࢬࣤࢪ໩ࡡ 㜴ᐐ࡚ࡢ࡝ࡂࠉDNA 々⿿㛜ጙ々ྙమᙟᠺࡡᢒโ࡚࠵ࡽࠉࣚ࢕ࢬࣤࢪ໩ᅄᏄࡡโ ᚒ࡛Mcm2-7 々ྙమࡡὩᛮ໩࠿ᐠ᥃࡞㏻ິࡊ࡙࠷ࡾྊ⬗ᛮ࠿♟ြࡈࡿࡒࠊ

(4)

➠஦❮ ᗆㄵ ➠ୌ⟿ DNA 々⿿ࣚ࢕ࢬࣤࢪ໩ᶭᵋ ┷ᰶ⏍∸ࡡࢣࢿ࣑ࡢ㛏ኬ࠾ࡗ々㞟࡚࠵ࡽࠉࡐࡡ々⿿ࢅୌᅂࡡ⣵⬂࿔᭿࡞ୌᗐ ࡓࡄḿ☔࡞㐑⾔ࡌࡾࡒࡴࠉ┷ᰶ⏍∸࡞ࡢኣᒪ࡞ࢂࡒࡾཚᐠ࡝โᚒᶭᵋ࠿ങ࠻ࡼ ࡿ࡙࠷ࡾࠊࡆࡡโᚒᶭᵋࡡ◒⥚ࡢࠉ㐿ఎ᝗ሒࡡን໩ࠉ⣵⬂ࡡṒࠉ࠵ࡾ࠷ࡢ⣵⬂ ࡡ⒬໩ࢅᘤࡀ㉫ࡆࡌࡆ࡛࠾ࡼࠉࡆࡡᶭᵋࡢࢣࢿ࣑ࡡᏭᏽᛮࢅྒྷࡾⅤ࡚ࠉ⏍࿤࡞ ࡛ࡖ࡙୘ྊḖ࡚࠵ࡾ࡛⩻࠻ࡼࡿࡾࠊ +LJ XU H )3& ࣚ࢕ࢬ ࣤࢪ໩ ௫ㄕ ࣚ࢕ࢬࣤࢪ໩௫ㄕࡢࠉ ᖳ࡞࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ࠾ࡼㄢ⿿ࡊࡒ᢫ฝᾦࢅ⏕࠷ࡒ↋⣵ ⬂ )3& 々⿿⣌࡞ࡻࡽᚋࡼࡿࡒᐁ㥺ࢅࡵ࡛࡞ᥞ၌ࡈࡿࡒࠊ┷ᰶ⣵⬂࡚ࡢ )3& 々⿿㛜ጙࢅセྊ ࡌࡾࣚ࢕ࢬࣤࢪ໩ᅄᏄ࠿Ꮛᅹࡊࠉ᯹Ⰵమ )3& ࡞⤎ྙࡌࡾࠊᰶහࡡࣚ࢕ࢬࣤࢪ໩ᅄᏄࡢ々⿿ 㛜ጙ࡞ࡻࡖ࡙୘Ὡᛮ໩ࡈࡿࡾ࠿ࠉ2 ᭿࡞࠽࠷࡙ᰶ⭯࠿ᓻቪࡌࡾ࡛ᰶአ࡞Ꮛᅹࡊ࡙࠷ࡾὩᛮᆵ ࡡࣚ࢕ࢬࣤࢪ໩ᅄᏄ࠿්ࡦ᯹Ⰵమ )3& ࡞⤎ྙ࡚ࡀࡾࡻ࠹࡞࡝ࡾࠊ

(5)

┷ᰶ⏍∸࠿ᬉ㐚Ⓩ࡞᭯ࡌࡾDNA 々⿿โᚒᶭᵋࡡୌࡗ࡛ࡊ࡙ DNA 々⿿ࣚ࢕ࢬ

ࣤࢪ໩ᶭᵋ࠿ᏋᅹࡊࠉDNA 々⿿ࡡ㛜ጙโᚒࡷ්々⿿ࡡ㜴Ḿ࡞ᶭ⬗ࡌࡾࡆ࡛࠿▩

ࡼࡿ࡙࠷ࡾ (Figure 1-1)(Blow and Laskey, 1988; Diffley, 1996; Tada and

Blow, 1998)ࠊ

ࢠ࣏ࣞࢲࣤ୕ࡡ々⿿㉫Ⅴ࡞࠽࠷࡙々⿿๑々ྙమ (pre-replication complex;

pre-RC) ࠿ᙟᠺࡈࡿࡾࡆ࡛࡞ࡻࡽ DNA 々⿿㛜ጙࡡࣚ࢕ࢬࣤࢪ໩࠿㉫ࡆࡾࠊ pre-RC ࡡᙟᠺ࡞ࡢᑛ࡝ࡂ࡛ࡵࠉ々⿿㉫Ⅴヾㆉ々ྙమ (ORC) (Chesnokov et al., 2001; Speck et al., 2005)ࠉCdc6 (Grallert and Sipiczki, 1991; Lisziewicz et al., 1988)ࠉCdt1 (Chong et al., 1995; Hofmann and Beach, 1994; Tada et al., 1999; Tada et al., 2001)ࠉMcm2-7 々ྙమ(Ishimi, 1997; Maine et al., 1984; You et al., 1999)ࡡ㸩⛸㢦ࡡࢰࣤࣂࢠ㈻࠿ᚪ㡪࡚࠵ࡽࠉࡆࡿࡼࡡࢰࣤࣂࢠ㈻࠿々⿿㉫Ⅴ

୕࡞㡨ḗ⤎ྙࡌࡾࡆ࡛࡚pre-RC ࡡᙟᠺ࠿㉫ࡆࡾ (Figure 1-2)(Bell and Dutta,

2002)ࠊ 㻲㻵㻦㻦㼇㼗㻔㼊㼈㼐㼌㼑㼌㼑㻰㻪㻔㻲㻵㻦㻦㼇㼆㻙㻦㼇㼗㻔㻲㻵㻦㻦㼇㼆㻙㻦㼇㼗㻔々⿿㉫Ⅴ 㻲㻵㻦㻦㼇㼆㻙㻦㼇㼗㻔㻰㻦㻰㻤㻷㻳ຊỀฦよ䜬䝑䝯䜲䞀 +LJ XU H )3& 々⿿ࣚ ࢕ࢬࣤ ࢪ໩ᶭ ᵋ

5UH7( ࡡᙟᠺࡢ 47( ࡡ々⿿㉫Ⅴ࡫ࡡ⤎ྙ࡞౪Ꮛࡊ࡙࠷ࡾࠊ47( ࡡ⤎ྙ࡞ࡻࡖ࡙ (IH ࡛ (IW

(6)

ࣚ࢕ࢬࣤࢪ໩ࡢࡱࡍ々⿿㉫Ⅴ࡫ࡡORC ࡡ⤎ྙ࡞ጙࡱࡽࠉORC ౪ᏋⓏ࡞ Cdc6

࡛ Cdt1 ࠿ࢠ࣏ࣞࢲࣤ୕࡫⤎ྙࡌࡾ(Coleman et al., 1996; Donovan et al.,

1997; Herbig et al., 1999; Maiorano et al., 2000)ࠊࡈࡼ࡞ Mcm2-7 々ྙమ࠿ Cdc6 ࡛ Cdt1 ࡞౪Ꮛࡊ࡙ࢠ࣏ࣞࢲࣤ୕࡫⤎ྙࡊ(Tanaka et al., 1997)ࠉCdc6

ࡡATP ຊỀฦよ࢙ࢾ࣭ࣜ࢟ࢅฺ⏕ࡊ࡙ࢠ࣏ࣞࢲࣤ࡞ᑙථࡈࡿࡾࡆ࡛࡚ࣚ࢕ࢬࣤ

ࢪ໩࠿Ᏸ஡ࡌࡾ(Figure 1-2)ࠊࡱࡒࠉࣚ࢕ࢬࣤࢪ໩㜴ᐐᅄᏄ࡛ࡊ࡙ geminin ࠿

ྜྷᏽࡈࡿ࡙࠽ࡽࠉCdt1 ࡛⤎ྙࡌࡾࡆ࡛࡞ࡻࡽࡐࡡᶭ⬗ࢅ㜴ᐐࡊࠉDNA 々⿿ࢅ

ᢒโⓏ࡞โᚒࡊ࡙࠷ࡾࡆ࡛࠿▩ࡼࡿ࡙࠷ࡾ(Lee et al., 2004; Tada et al., 2001; Wohlschlegel et al., 2000)ࠊ ➠஦⟿ ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ᢫ฝᾦ↋⣵⬂ᐁ㥺⣌ ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ᢫ฝᾦ↋⣵⬂ᐁ㥺⣌࡚ࡢࠉ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ࠾ࡼ ᚋࡒ᢫ฝᾦ࡫㞕ࡡ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜࡻࡽㄢ⿿ࡊࡒ⢥Ꮔᰶ )3& ࢅຊ࠻ࡾࡆ࡛࡚ࠉ ࢠ࣏ࣞࢲࣤࡡ⬲ฆ㞗࠾ࡼDNA 々⿿ࠉ᯹Ⰵమࡡฆ㞗࣬ฦ㒼ࡱ࡚ࡡ⣵⬂࿔᭿࡞ఔ࠹

ୌ㏻ࡡࢱ࢕ࢻ࣐ࢴࢠ࡝ን໩ࢅ↋⣵⬂Ⓩ࡞්⌟࡚ࡀࡾ (Figure 1-3) (Lohka and Masui, 1983)ࠊ↋⣵⬂࡚࠵ࡾ࠿ࡹ࠻ࠉ⣵⬂࡚ࡢ⮬Ṓ࡛࡝ࡾ᮪௲ୖ࡚ࡵᐁ㥺ࢅ⾔ ࠹ࡆ࡛࠿࡚ࡀࠉࡈࡼ࡞ඞ␷㝎ཡ࡝࡜ࡡ≁␏Ⓩᢘమࢅ⏕࠷ࡒ⏍໩ᏕⓏᡥἪࢅᐖ᪾ ࡞ᑙථࡌࡾࡆ࡛࠿࡚ࡀࡾࡒࡴࠉࣚ࢕ࢬࣤࢪ໩ࢅྱࡴࡒ⣵⬂࿔᭿ࡡ々㞟࡝ཬᚺ࡞ 㛭ࢂࡾࢰࣤࣂࢠ㈻ࡡᶭ⬗ࡡよᯊࢅ㎷㏷࠾ࡗ᪺▉࡞⾔࠹ࡆ࡛࠿࡚ࡀࡾࠊ

(7)

+LJ XU H ࢓ࣆࣛ ࢜ࢵ࣒ ࢙࢝ࣜ ༵᢫ฝ ᾦ↋⣵ ⬂ᐁ㥺⣌ ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜࡡ༵ࢅ㐪ᚨ◒◃ࡊ࡙ᚋࡼࡿࡒ༵᢫ฝᾦ࡞ᰶ⭯ࢅ㝎ཡࡊࡒ⢥Ꮔᰶ )3& ࢅຊ࠻ࡾࡆ࡛࡞ࡻࡽࠉิ᭿⫿࡚ふᐳࡈࡿࡾୌ㏻ࡡ⣵⬂࿔᭿ࡡ㐅⾔ࢅモ㥺⟮හ࡚ᐖ᪾࡞්⌟ ࡌࡾࡆ࡛࠿࡚ࡀࡾࠊ ➠୔⟿ ᮇ◂✪ࡡ┘Ⓩ Cdt1 ࡢࠔMcm2-7 々ྙమࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞さịࡈࡿࡾࠕ࡛࠷࠹▩ぜ௧୕࡞ ⌦よࡈࡿ࡙࠷ࡾ࡛ࡢゕ࠷㞬ࡂࠉࡐࡡ⏍໩ᏕⓏὩᛮࡷฦᏄⓏ࡝ᶭ⬗࡞ࡗ࠷࡙୘᪺ ࡝Ⅴ࠿ኣ࠷ࠊCdt1 ࡢࠉ࢓࣐ࢿ㓗ୌḗ㒼า୯࡞ࡵ᪜Ꮛࡡ㓕⣪Ὡᛮࢅ♟ࡌࡻ࠹࡝࣓ ࢲ࣭ࣆࡢぜࡗ࠾ࡖ࡙࠽ࡼࡍࠉ௙ࡡࢰࣤࣂࢠ㈻࡛┞பష⏕ࢅ⾔࠹ࡆ࡛࡚ࡐࡡᙲ๪ ࢅᯕࡒࡊ࡙࠷ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾࠊ

(8)

㻲㻵㻦㻲㻵㻦㻦㼇㼆㻙㻲㻵㻦㻦㼇㼗㻔㻲㻵㻦㻦㼇㼆㻙㻦㼇㼗㻔㻲㻵㻦㻦㼇㼗㻔㻦㼇㼆㻙㻲㻵㻦㻦㼇㼆㻙㻦㼇㼗㻔㻰㻦㻰㻦㼇㼗㻔䛮㻦㼇㼆㻙䛴 ┞பష⏕䠑㻦㼇㼗㻔䛮㻦㼇㼆㻙䛴相互作用なし？䝭䜨䜿䝷䜽໩䛴Ᏸ஡ +LJ XU H (IW ࡛ ( IH ࡡࢠ ࣏ࣞࢲ ࣤ⤎ྙ ࡞ ࠽ࡄࡾ㡨 ᗆ

(IW ࡛ (IH ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡢ㡨ᗆ࠿Ꮛᅹࡊࠉ(IH ࠿඙࡞ࢠ࣏ࣞࢲࣤ࡞⤎ྙࡊ࡝ࡄ ࡿࡣ࡝ࡼ࡝࠷ࠊࡆࡡ㡨ᗆ࠿஗ࡿࡒ㝷࡞ࡢࠉࣚ࢕ࢬࣤࢪ໩ࡢᏰ஡ࡊ࡝࠷ࠊ ࡆࡿࡱ࡚ᙔ◂✪ᐄ࡞࠽࠷࡙ࠉCdt1 ࠿ Cdc6 ࡞඙ࢆࡋ࡙ࢠ࣏ࣞࢲࣤ⤎ྙࡊ࡙ࡵ Mcm2-7 々ྙమࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ᶭ⬗࡚ࡀ࡝࠷ࡆ࡛࠿♟ࡈࡿ࡙࠷ࡾ(Figure 1-4) (Tsuyama et al., 2005)ࠊࡆࡡࡆ࡛࠾ࡼ Cdt1 ࡛ Cdc6 ࡞రࡼ࠾ࡡ┞பష⏕࠿ Ꮛᅹࡊࠉ༝ㄢⓏ࡞ᶭ⬗ࡊ࡙࠷ࡾྊ⬗ᛮ࠿⩻࠻ࡼࡿࡾࠊ ࡐࡆ࡚ࠉᮇ◂✪࡚ࡢࠉCdt1 ࡡᶭ⬗㡷ᇡ᩷∞ࢅྱࡳ⤄ᥦ࠻ࢰࣤࣂࢠ㈻ࢅ⏕࠷࡙ࠉ Cdt1 ࡡ Cdc6 ࡛ࡡᶭ⬗Ⓩ┞பష⏕ࡡよᯊࢅ⾔࠷ࠉࡱࡒࡆࡡ᩷∞໩ࡊࡒ Cdt1 ࠿ ࣚ࢕ࢬࣤࢪ໩ࡷ DNA 々⿿࡞࡜ࡡࡻ࠹࡝ᙫ㡢ࢅ୙࠻ࡾࡡ࠾よ᪺ࡌࡾࡆ࡛ࢅ┘Ⓩ ࡛ࡊࡒࠊ

(9)

➠ୌ⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡష⿿ Cdt1 ࡡᶭ⬗㡷ᇡࡢ N ᮆ❻ഁ (Cdt1-N; 1-243 a.a)ࠉ୯ኳ (Cdt1-M; 243-447 a.a)ࠉC ᮆ❻ഁ (Cdt1-C; 447-621 a.a)࡛ኬࡀࡂ୔ࡗ࡞ฦࡄࡾࡆ࡛࠿࡚ࡀࡾ (Figure 3-1)ࠊ !"#$ %& !"#$ %' !"#$ %! ()* +, -.* +, /01 !""# &2 -34*56789: !"#$ !"#$ %& !"#$ %' !"#$ %! -;( ;;) <-$ $ $%&'('()'(*'($+ 59=7>:9>?*6=#989#@ '!'*A9>"9>? BCD#7D5@:9: /E1 /64*61 F -F ;F <F GF $FF > D> 7 ,&E* :@>#H7:9: /I* DJ* =D>#CD51 29:%!"#$ 29:%!"#$ %& 29:%!"#$ %' 29:%!"#$ %! /!1 +LJ XU H (IW ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ ష⿿ &࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ (IW ࡡᶭ⬗ࢺ࣒࢕ࣤ '(IW ᩷∞ࢰࣤࣂࢠ㈻ -LV ࢰࢡࢰࣤࣂࢠ㈻࡛ࡊ࡙ష⿿࣬⢥⿿ࡊࡒࠊ

((IW ࢅඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ୯࡚ࡡ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ )3& 々⿿Ὡᛮ (IW ࢅඞ␷

㝎ཡࡊࡒ༵᢫ฝᾦ࡞ࠉ_{5I&95ࠉ-LV(IW3ࠉ2ࠉ(⤂⃨ᗐ Q2ࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ} ᮇµ1ࢅ῟ຊࡊࠉΥ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊ ฦᚃࠉ)3& ྙᠺ㔖ࢅῼᏽࡊࠉඞ␷ ๑⾉ΰ࡚ඞ␷㝎ཡ࡛ྜྷᵕࡡฌ⌦ࢅࡊࡒ༵᢫ฝᾦ࡞ࠉࢤࣤࢹ࣭ࣞࣜ GXIIHU ࢅ῟ຊࡊࡒ༵᢫ฝ ᾦ࡞࠽ࡄࡾ )3& ྙᠺ㔖ࢅ ࡛ࡊ࡙ )3& 々⿿ὩᛮࢅẒ㍉ࡊࡒࠊ Cdt1-N ࡢ Cdt1 ࡡฦよ➴ࡡᶭ⬗โᚒ࡞㛭ࢂࡽࠉCdt1-M ࡢ㜴ᐐࢰࣤࣂࢠ㈻࡚

(10)

࠵ࡾ geminin ࡛ࡡ⤎ྙ࡞ࠉCdt1-C ࡢ Mcm2-7 々ྙమ࡛ࡡ⤎ྙ࡞ᐞ୙ࡌࡾࡆ࡛ ࠿ሒ࿈ࡈࡿ࡙࠷ࡾ (Ferenbach et al., 2005)ࠊࡱࡒ Cdt1-N ࡡ㡷ᇡࢅḖ࠷࡙ࡵࠉ ࣚ࢕ࢬࣤࢪ໩Ὡᛮࢅࡵࡗࡆ࡛࠿▩ࡼࡿ࡙࠷ࡾ (Figure 3-1 A)ࠊࡈࡼ࡞࣏ࢗࢪ Cdt1 ࡞࠽࠷࡙ࠉCdt1-N ࠾ࡼ Cdt1-M ࡡ㡷ᇡ (1-363 a.a) ࡞࠾ࡄ࡙ࠉDNA ࡛ࡡ ⤎ྙ⬗ࢅ᭯ࡌࡾ࡛࠷࠹ࡆ࡛࠿ሒ࿈ࡈࡿ࡙࠷ࡾ (Yanagi et al., 2002)ࠊCdt1-Mࠉ Cdt1-C ࡢ㧏➴┷ᰶ⏍∸࡞࠽࠷࡙㧏ᗐ࡞ಕᏋࡈࡿ࡙࠽ࡽࠉCdt1 ࡡᶭ⬗࡞࠽࠷࡙ 㟸ᖏ࡞㔔さ࡝㡷ᇡ࡚࠵ࡾࡆ࡛࠿஢᝷ࡈࡿࡾࠊ ࡐࡆ࡚ࡈࡼ࡝ࡾ Cdt1 ࡡᶭ⬗よᯊࢅ⾔࠹ࡆ࡛ࢅ┘Ⓩ࡞ࠉCdt1-NࠉCdt1-Mࠉ Cdt1-C ࢅ N ᮆ❻࡞ His ࢰࢡࢅ௛୙ࡊࡒࢰࣤࣂࢠ㈻࡛ࡊ࡙Ⓠ⌟࣬⢥⿿ࡊࡒ (Figure 3-1 B)ࠊහᅄᛮ Cdt1 ࢅඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ୯࡞ࡆࡿࡼ᩷∞໩ࡊࡒ Cdt1 ࢅ῟ຊࡊࡒࡡࡔ࡞ DNA 々⿿Ὡᛮࢅῼᏽࡊࠉࡆࡿࡼࡡ᩷∞ࢰࣤࣂࢠ㈻ࡢ DNA 々⿿Ὡᛮࢅᣚࡒ࡝࠷ࡆ࡛ࢅ☔ヾࡊࡒ (Figure 3-1 C)ࠊ ➠஦⟿ Cdt1 ࡡ Cdc6 ࡛ࡡ⤎ྙ㒂నࡡྜྷᏽ Cdt1 ࡡ࡜ࡡ㒂న࡚ Cdc6 ࡛⤎ྙࡌࡾࡡ࠾᪺ࡼ࠾࡛ࡌࡾࡒࡴ࡞ࠉCdt1 ᩷∞ࢰࣤ ࣂࢠ㈻(His-Cdt1ࠉHis-Cdt1-NࠉHis-Cdt1-MࠉHis-Cdt1-C) ࡛ GST-Cdc6 ࢅ

⏕࠷࡙ࠉpull-down assay ࢅ⾔ࡖࡒ (Figure 3-2)ࠊ྘⛸ Cdt1 ࠿ථࡖࡒ buffer ୯

࡞ GST-Cdc6 ࢅ῟ຊࡊࠉ23Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃ࡞ࠉNi-NTA ࣑࡛࢜ࣚ⤎

ྙࡌࡾࢰࣤࣂࢠ㈻ࢅࢗ࢘ࢪࢰࣤࣇࣞࢴࢹἪ࡞ࡻࡽ᳠ฝࡊࡒࠊࡱࡍࠉHis-Cdt1 (ධ

㛏) ࢅ⏕࠷࡙᳠ゞࡊࡒ⤎ᯕࠉCdt1 ࠿ Cdc6 ࡛⤎ྙࡌࡾࡆ࡛࠿☔ヾࡈࡿࡒ(Figure

3-2)ࠊࡈࡼ࡞ࠉHis-Cdt1-M ࠿ GST-Cdc6 ࡛⤎ྙࡌࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖࡒ

(11)

㻪㻶㻷㻐㻦㼇㼆㻙㻎㻫㼌㼖㻐㻦㼇㼗㻔㻃㼙㼄㼕㼌㼄㼑㼗㻋㼉㼘㼏㼏㻃㼏㼈㼑㼊㼗㼋㻏㻃㻱㻏㻃㻰㻏㻃㻦㻌㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻱㼌㻐㻱㻷㻤 wash 㻉㻃㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃㼅㼌㼑㼇㼌㼑㼊㻃㼓㼕㼒㼗㼈㼌㼑㼖㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻎㻎㻎㻎㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻎㻎㻎㻎㼓㼘㼏㼏㻃㼇㼒㼚㼑 input 㻪㻶㻷㻐㻦㼇㼆㻙㼉㼘㼏㼏㻐㼏㼈㼑㼊㼗㼋㻱㻰㻃㼒㼕㻃㻦 +LJ XU H SXO O IR \Q F VVF ^ ( IW ࡛ ( IH ࡡ⤎ྙ䐖

µ2 ࡡ ,89(IH ࡛ µ2 ࡡ -LV(IW ᩷∞(IW3ࠉ(IW2ࠉ(IW(ࢅ Υ࡚ ฦ㛣 ࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ3L39& ࢅ⏕࠷࡙ -LV(IW ᩷∞࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈻ࢅ᳠ฝࡊࡒࠊ

(IWࠉ(IW ᩷∞ࢰࣤࣂࢠ㈻ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘమࠉ(IH ࡡྜྷᏽ࡞ࡢᢘ =HQRSXV(IH

ᢘమࢅ⏕࠷ࡒࠊ

ࡗࡁ࡞ࠉGSTࠉGST-Cdc6 ࢅྱࡳ buffer ୯࡞ࠉHis-Cdt1 ࢅ῟ຊࡊࠉྜྷᵕࡡ᪁

Ἢ࡚ࠉpull-down assay ࢅ⾔ࡖࡒ (Figure 3-3 A)ࠊHis-Cdt1 ࡢ GST-Cdc6 ࡛ࡡ

ࡲ⤎ྙࡊࠉGST ࡛ࡢ⤎ྙࡊ࡝࠾ࡖࡒࡆ࡛࠾ࡼࠉHis-Cdt1 ࡛ GST-Cdc6 ࡡ⤎ྙ

ࡢࠉCdt1 ࡛ GST ࢰࢡ࡛ࡡ┞பష⏕࡚ࡢ࡝ࡂࠉCdt1 ࡛ Cdc6 ࡡ⤎ྙ࡞㉫ᅄࡌࡾ

ࡆ࡛࠿☔ヾࡈࡿࡒ(Figure 3-3 A)ࠊ

ࡈࡼ࡞ATP Ꮛᅹୖࠉ㟸Ꮛᅹୖ ࡐࡿࡑࡿ࡞ࡗ࠷࡙ His-Cdt1ࠉGST-Cdc6 ࢅ῟

ຊࡊࠉྜྷᵕ࡞pull-down assay ࢅ⾔ࡖࡒ (Figure 3-3 B)ࠊATP 㟸Ꮛᅹୖ࡚ࡢ ATP

Ꮛᅹୖ࡞Ẓ࡬࡙ⱕᖱ఩ୖࡊࡒࡵࡡࡡࠉ୦⩽ࡡ⤎ྙ࠿☔ヾࡈࡿࡒ(Figure 3-3 B)ࠊ

ࡻࡖ࡙ Cdt1 ࡛ Cdc6 ࡡ⤎ྙࡢࠉᚪࡍࡊࡵ ATP ࢅᚪさ࡛ࡢࡊ࡝࠷ࡆ࡛࠿♟ြࡈ

(12)

㼓㼘㼏㼏㻃㼇㼒㼚㼑㻃㻞㻃㻱㼌㻐㻱㻷㻤㼌㼑㼓㼘㼗㻎㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻪㻶㻷㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻎㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻪㻶㻷㻫㼌㼖㻐㻦㼇㼗㻔㻎㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻱㼌㻐㻱㻷㻤㼚㼄㼖㼋㻃㻉㻃㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃 binding proteins 㻪㻶㻷㻃㼒㼕㻃㻪㻶㻷㻐㻦㼇㼆㻙 * * 㼌㼑㼓㼘㼗㻎㻎㻐㻐㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻤㻷㻳㻎㻎㻐㻐㻎㻎㻎㻎㻎㻎㻎㻎㻎㻎㻐㻐㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻪㻶㻷㻐㻦㼇㼆㻙㻎㻫㼌㼖㻐㻦㼇㼗㻔㻋㻃㻎㻒㻐㻤㻷㻳㻃㻌㻎㻎㻐㻐㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻱㼌㻐㻱㻷㻤㼚㼄㼖㼋㻃㻉㻃㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃 binding proteins 㼓㼘㼏㼏㻃㼇㼒㼚㼑㻃㻞㻃㻱㼌㻐㻱㻷㻤 A B +LJ XU H SXO O IR \Q F VVF ^ ( IW ࡛ ( IH ࡡ⤎ྙղ

&(IW ࡛ (IH ࡡ⤎ྙ࡞࠽ࡄࡾ ,89 ࢰࢡࡡᙫ㡢 µ2 ࡡ ,89(IH ࡵࡊࡂࡢ ,89 ࢅྱࡳࣆ ࣚࢠࢨࣘࣤ࡞ࠉ µ2 ࡡ -LV(IW ࢅ῟ຊࡊࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ3L39&

ࢅ⏕࠷࡙ -LV(IW ࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈻ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘ

మࠉ(IH ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IH ᢘమࠉ,89 ࡡྜྷᏽ࡞ࡢᢘ ,89 ᢘమࢅ⏕࠷ࡒࠊ

'(IW ࡛ (IH ࡡ⤎ྙ࡞࠽ࡄࡾ &95 ࡡᙫ㡢 µ2 ࡡ ,89(IH ࡛ µ2 ࡡ -LV(IW ࢅ &95 ῟ຊ࣬㟸῟ຊ᮪௲ୖ࡞࠽࠷࡙ࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ3L39& ࢅ⏕࠷࡙

-LV(IW ࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈻ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ =HQRSXV(IW ᢘమࠉ(IH

ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IH ᢘమࢅ⏕࠷ࡒࠊ

➠୔⟿ Cdt1-Cdc6 ┞பష⏕࡞ᑊࡌࡾ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡ❿ྙ

ࡈࡼ࡞ࠉCdc6 ⤎ྙ⬗ࢅࡵࡗ Cdt1-M ࠿ Cdt1 ࡛ Cdc6 ࡡ┞பష⏕࡞❿ྙ࡚ࡀ

ࡾ࠾࡞ࡗ࠷࡙ pull-down assay ࡞ࡻࡽ᳠ゞࢅ⾔ࡖࡒ (Figure 3-4)ࠊୌᏽ㔖ࡡ

(13)

࢘ࢪࢰࣤࣇࣞࢴࢹἪ࡞ࡻࡽ᳠ฝࡊࡒࠊࡐࡡ⤎ᯕࠉHis-Cdt1-M ࡡ⃨ᗐࡡቌຊ࡛࡛ ࡵ࡞ࠉGST-Cdc6 ࡫ࡡ His-Cdt1-M ࡡ⤎ྙ㔖࠿ቌຊࡊࠉୌ᪁࡚ His-Cdt1 ࡡ GST-Cdc6 ࡫ࡡ⤎ྙ㔖࠿఩ୖࡌࡾࡆ࡛࠿☔ヾࡈࡿࡒ(Figure 3-4)ࠊࡻࡖ࡙ࠉ Cdt1-M ࠿ Cdt1 ࡡ Cdc6 ࡛ࡡ⤎ྙ࡞❿ྙࡌࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖࡒࠊ 㻪㻶㻷㻐㻦㼇㼆㻙㻃㻋㻃㻓㻑㻔䃒㻰㻃㻌㻎㻫㼌㼖㻐㻦㼇㼗㻔㻃㻋㻃㻓㻑㻔䃒㻰㻃㻌㻎㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻪㼏㼘㼗㼄㼗㼋㼌㼒㼑㼈㼖㼈㼓㼋㼄㼕㼒㼖㼈㼚㼄㼖㼋㻃㻉㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃㼅㼌㼑㼇㼌㼑㼊㼓㼕㼒㼗㼈㼌㼑㼖㻪㻶㻷㻐㻦㼇㼆㻙㻫㼌㼖㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㼓㼘㼏㼏㻃㼇㼒㼚㼑 input 㻓㻑㻙㻓㻑㻖㻓㻑㻔㻘㻓㻑㻓㻚㻘㻓㻓㻓㻑㻓㻚㻘㻓㻑㻔㻘㻓㻑㻖㻓㻑㻙㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㼆㼒㼑㼆㻑㻋䃒㻰㻃㻌 +LJ XU H (IW (I H ┞ பష⏕ ࡞ᑊࡌ ࡾ (I W ᩷∞ࢰ ࣤࣂࢠ ㈻ࡡ❿ ྙᛮ µ2 ࡡ ,89(IH ࡛ µ2 ࡡ -LV(IW ࡞྘⃨ᗐ㸝 µ2ࠉ µ2ࠉ µ2ࠉ µ2㸞 ࡡ -LV(IW2 ࢅ῟ຊࡊࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ,OXWFWKLRQH8HSKFURVH ࢅ

⏕࠷࡙ ,89(IH ࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈻ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘమࠉ

(IH ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IH ᢘమࠉ(IW2 ࡡྜྷᏽ࡞ࡢᢘ +1&, ᢘమࢅ⏕࠷ࡒࠊ

(14)

➠ᄿ⟿ Cdt1-Cdt1 ࢰࣤࣂࢠ㈻㛣┞பష⏕

Cdt1 ࡡධ㛏ࡢ⣑ 70 kDa ࡚࠵ࡾ࠿ࠉࢣࣜࢀ㐛ࢅ⾔࠹࡛ 300 kDa ௛㎾ࡡన⨠࡞ Cdt1 ࠿⁈ฝࡈࡿ࡙ࡂࡾࡆ࡛࠾ࡼࠉCdt1 ࡢኣ㔖మᙟᠺ⬗ࢅᣚࡗྊ⬗ᛮ࠿♟ြࡈ ࡿ࡙࠷ࡒࠊ

ࡐ ࡆ ࡚ ࠉGST-His-Cdt1 ࡛ His-Cdt1 ࢅ Ί ྙ ࣬ ࢕ ࣤ ࢞ ࣖ ࣊ ࣭ ࢹ ࡊ ࠉ

Gultathione-Sepharose ࢅ⏕࠷ࡒ pull-down assay ࡞ࡻࡽࠉ୦⩽࠿⤎ྙࡊᚋࡾ࠾ ᳠ゞࢅ⾔ࡖࡒ(Figure 3-5)ࠊ 㼓㼘㼏㼏㻃㼇㼒㼚㼑㻎㻐㻪㻶㻷㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻎㻎㻎㻎㻪㻶㻷㻐㻦㼇㼗㻔㻎㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻪㼏㼘㼗㼄㼗㼋㼌㼒㼑㼈㼖㼈㼓㼋㼄㼕㼒㼖㼈㼚㼄㼖㼋㻃㻉㻃㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃 binding proteins 㻫㼌㼖㻐㻦㼇㼗㻔㼌㼑㼓㼘㼗㻎㻐㻎㻎㻎㻎㻋㻎㻃㻫㼌㼖㻐㼊㼈㼐㼌㼑㼌㼑㻌㻫㼌㼖㻐㼊㼈㼐㼌㼑㼌㼑㼊㼈㼐㼌㼑㼌㼑㻎㻐㻐㻐㻐㻎㻦㼇㼗㻔㻪㻶㻷㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔 +LJ XU H SXO O IR \Q F VVF ^ ( IW (I W ࢰ ࣤࣂࢠ㈻㛣┞ப ష⏕ µ2 ࡡ ,89(IW ࢅྱࡳࣆࣚࢠࢨࣘࣤ࡞ µ2 ࡡ -LV(IWࠉ-LVJHPLQLQ ࢅ῟ຊࡊࠉΥ ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ,XOWFWKLRQH8HSKFURVH ࢅ⏕࠷࡙ ,89(IW ࡛⤎ྙࡌࡾࢰ ࣤࣂࢠ㈻ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘమࠉJHPLQLQ ࡡྜྷᏽ࡞ࡢᢘ =HQRSXV JHPLQLQ ᢘమࢅ⏕࠷ࡒࠊ

(15)

ࡐࡡ⤎ᯕࠉGST-His-Cdt1 ࡞ His-Cdt1 ࠿⤎ྙࡌࡾࡆ࡛࠿☔ヾࡈࡿࡒ (Figure 3-5)ࠊࡱࡒࠉࡆࡆ࡞0.1 µM ࡡ His-geminin ࢅ῟ຊࡊ࡙ࡵ GST-Cdt1 ࡫ࡡ His-Cdt1

ࡡ⤎ྙ㔖ࡢን໩ࡊ࡝࠾ࡖࡒ (Figure 3-5)ࠊࡻࡖ࡙ࠉCdt1 ࡢኣ㔖మᙟᠺ⬗ࢅᣚࡔࠉ

ࡱࡒࡐࡡ⤎ྙ࡞geminin ࡢ❿ྙࡊ࡝࠷ࡆ࡛࠿♟ြࡈࡿࡒࠊ

ḗ࡞Cdt1 ࡡኣ㔖మᙟᠺ࠿࡜ࡡ㒂నࢅ௒ࡊ࡙⾔ࢂࡿ࡙࠷ࡾࡡ࠾᪺ࡼ࠾࡛ࡌࡾ

ࡒࡴࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ⏕࠷࡙ pull-down assay ࢅ⾔ࡖࡒ (Figure 3-6)ࠊ

ࡐࡡ⤎ᯕࠉHis-Cdt1-M ࠿ GST-Cdt1 ࡛ᙁࡂ⤎ྙࡊ࡙࠽ࡽࠉHis-Cdt1-Nࠉ His-Cdt1-C ࡵⱕᖱ࡚ࡢ࠵ࡾ࠿ࠉGST-Cdt1 ࡛⤎ྙࡊ࡙࠷ࡒ (Figure 3-6)ࠊࡆࡿ ࡻࡽࠉCdt1 ࡡኣ㔖మᙟᠺࡢ Cdt1-M ࢅ୯ᚨ࡞⾔ࢂࡿ࡙࠷ࡾࡆ࡛࠿᪺ࡼ࠾࡛࡝ࡖ ࡒࠊ 㻪㻶㻷㻐㻦㼇㼗㻔㻎㻫㼌㼖㻐㻦㼇㼗㻔㻃㼙㼄㼕㼌㼄㼑㼗㻋㼉㼘㼏㼏㻃㼏㼈㼑㼊㼗㼋㻏㻃㻱㻏㻃㻰㻏㻃㻦㻌㼌㼑㼆㼘㼅㼄㼗㼌㼒㼑㻪㼘㼏㼗㼄㼗㼋㼌㼒㼑㼈㼖㼈㼓㼋㼄㼕㼒㼖㼈㼚㼄㼖㼋㻃㻉㻃㼈㼏㼘㼗㼌㼒㼑㼇㼈㼗㼈㼆㼗㼌㼒㼑㻃㼒㼉㻃 binding proteins 㻪㻶㻷㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻎㻎㻎㻎㻎㻎㻎㻎㻎㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻐㻎㻎㻎㻎㼓㼘㼏㼏㻃㼇㼒㼚㼑㼌㼑㼓㼘㼗㻪㻶㻷㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦 +LJ XU H SXO O IR \Q F VVF ^ ( IW ࡛ ( IW ࡡ┞பష ⏕㒂న ࡡ≁ᏽ

µ2 ࡡ ,89(IW ࡛ µ2 ࡡ -LV(IW ᩷∞(IW3ࠉ(IW2ࠉ(IW(ࢅ Υ࡚ ฦ㛣 ࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ,XOWFWKLRQH8HSKFURVH ࢅ⏕࠷࡙ ,89(IW ࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈻

ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘమࠉ(IW ᩷∞ࢰࣤࣂࢠ㈻ࡡྜྷᏽ࡞ࡢᢘ +1&,

(16)

➠ᄿ❮ ༵᢫ฝᾦ࡚ࡡ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ⏕࠷ࡒよᯊ ➠ୌ⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻࡞ࡻࡾහᅄᛮ Cdt1 ࡡฦよᢒโ Cdt1 ࡡࢰࣤࣂࢠ㈻ฦよࢅ௒ࡊࡒㄢ⟿࡞ࡢ Skp1-Cul1-F-box (SCF)-Skp2 ࠽ࡻ ࡦ Cul4-DDB1 ࡡ஦⛸ࡡ␏࡝ࡾࣗࣄ࢞ࢲ࣭ࣤࣛ࢝ࢭ࠿㛭୙ࡌࡾ⤊㊪࠿Ꮛᅹࡌࡾ ࡆ ࡛ ࠿ ሒ ࿈ ࡈ ࡿ ࡙ ࠷ ࡾ ࠊS ᭿࡞࠽࠷࡙ Cdt1 ࡢ N ᮆ❻㡷ᇡ࡞Ꮛᅹࡌࡾ

proliferating cell nuclear antigen (PCNA) ⤎ྙ࣓ࢲ࣭ࣆ (PIP box ; 6-13 a.a)

ࢅ௒ࡊࡒPCNA ࡛ࡡ⤎ྙ࡞౪ᏋⓏࡊ࡙ Cul4-DDB1 ࡞ࡻࡾฦよࢅུࡄࡾ (Arias

and Walter, 2006; Maga and Hubscher, 2003)ࠊୌ᪁ࠉࣃࢹ࡞࠽࠷࡙ Cdt1 ࡢ Cul4-DDB1 ࡡ௙࡞ࠉcyclin- dependent kinase (CDK) ࡞ࡻࡾࣛࣤ㓗໩࡞౪Ꮛࡊ

࡙ SCF-Skp2 ࡞ࡻࡾฦよࢅུࡄࡾࡆ࡛࠿ሒ࿈ࡈࡿ࡙࠷ࡾ (Nishitani et al., 2006)ࠊࡆࡡࡒࡴࠉࣃࢹ⣵⬂࡚ࡢ஦⛸ࡡࣗࣄ࢞ࢲ࣭ࣤࣛ࢝ࢭ࠿ Cdt1 ࡡฦよ࡞഼ ࠷࡙࠷ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾࠊᮇ◂✪࡚౐⏕ࡊ࡙࠷ࡾ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ᢫ฝ ᾦ↋⣵⬂ᐁ㥺⣌࡞࠽࠷࡙ࡢࠉCdt1 ࡡ N ᮆ❻ഁ࠿ࢰࣤࣂࢠ㈻ฦよࡡࢰ࣭ࢣࢴࢹ࡞ ࡝ࡖ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡾࠊࡐࡆ࡚ࠉHis-Cdt1-N ࢅ㐛๨࡞῟ຊࡊࡒ㝷࡞ࠉහᅄᛮ Cdt1 ࡡฦよࢅᢒ࠻ࡾࡆ࡛࠿࡚ࡀࡾ࠾᳠ゞࡊࡒࠊ ༵᢫ฝᾦ࡞His-Cdt1-N ࢅ῟ຊࡊࠉ23Υ࡚ 60 ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉࢗ ࢘ࢪࢰࣤࣇࣞࢴࢹ࡞ࡻࡽCdt1 ࡡฦよ࡫ࡡᙫ㡢ࢅㄢ࡬ࡒ(Figure 4-1)ࠊ఩⃨ᗐࡡ His-Cdt1-N ࡚ࡢ࡮࡛ࢆ࡜ን໩ࡢぜࡼࡿ࡝࠾ࡖࡒ࠿ࠉ⤂⃨ᗐ 50 µM ࡞࡝ࡾࡻ࠹ ࡞His-Cdt1-N ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡚ࡢࠉ↋῟ຊࡡ༵᢫ฝᾦ࡞Ẓ࡬හᅄᛮ Cdt1 ࡡฦよ࠿ᢒ࠻ࡼࡿ࡙࠷ࡒ (Figure 4-1)ࠊࡆࡿࡻࡽ㧏⃨ᗐ His-Cdt1-N ࡢහᅄᛮ Cdt1 ࡡฦよࢅᢒโࡌࡾࡆ࡛࠿ྊ⬗࡚࠵ࡾࡆ࡛࠿♟ࡈࡿࡒࠊ

(17)

㼈㼑㼇㼒㻑㻃㻦㼇㼗㻔㻓㻙㻓㻋㼐㼌㼑㻑㻌㻘㻓㻔㻕㻑㻘㻖㻑㻔㻓㻑㻚㻘㻓㻑㻔㻜㻓㻑㻓㻘㻓㻓㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻋䃒㻰㻌㻨㼛㼗㼕㼄㼆㼗㼖㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻓㻙㻓㻋㼐㼌㼑㻑㻌 +LJ XU H (IW 3 ࡡහ ᅄᛮ ( IW ࡡฦよ ᢒโ ༵᢫ฝᾦ࡞⾪♟ࡡ⤂⃨ᗐࡡ -LV(IW3 ࢅ῟ຊࡊࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ

ࢗ࢘ࢪࢰࣤࣇࣞࢴࢹἪ࡞ࡻࡽࠉ༵᢫ฝᾦ୯ (IW ࢅ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW

ᢘమࢅ⏕࠷ࡒࠊ ➠஦⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙ Cdt1 ࠿௙ࡡࣚ࢕ࢬࣤࢪ໩ᅄᏄྜྷᵕࢠ࣏ࣞࢲࣤ୕࡫⤎ྙࡌࡾࡆ࡛ࠉDNA ࡛ࡡ ⤎ྙὩᛮࢅ᭯ࡊ࡙࠷ࡾࡆ࡛࠿ࡆࡿࡱ࡚࡞ሒ࿈ࡈࡿ࡙࠷ࡾ(Yanagi et al., 2002)ࠊ ࡊ࠾ࡊࠉCdt1 ࡡ DNA ࠵ࡾ࠷ࡢࢠ࣏ࣞࢲࣤ⤎ྙὩᛮ࡛ᶭ⬗ࡡ㛭㏻ᛮ࡞ࡗ࠷࡙ࡢ ୘᪺࡝Ⅴ࠿ኣ࠷ࠊ ࡐࡆ࡚ࡱࡍࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻࠿ࢠ࣏ࣞࢲࣤ୕࡫⤎ྙࡊᚋࡾ࠾᳠ゞࡊࡒ (Figure 4-2)ࠊCdt1 ࢅඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ࡞ࠉ⢥Ꮔᰶ DNAࠉCdt1 ᩷∞ࢰࣤ ࣂࢠ㈻ࢅ῟ຊࡊࠉ23Υ࡚ 20 ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒ㝷ࡡ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡗ࠷࡙☔ヾࡊࡒࠊࡐࡡ⤎ᯕࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡢ༟≺࡚ ࡐࡿࡑࡿࢠ࣏ࣞࢲࣤ࡞⤎ྙࡌࡾࡆ࡛࠿࡚ࡀࡒ (Figure 4-2)ࠊࡊ࠾ࡊ࡝࠿ࡼࠉࡐ

(18)

ࡡ⤎ྙ㔖ࡢࠉඞ␷๑⾉ΰࢅ⏕࠷࡙ඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ࡛Cdt1 ࢅඞ␷㝎ཡࡊࡒ ༵᢫ฝᾦ࡛࡚ࡢኬࡀ࡝㐢࠷ࡢぜࡼࡿ࡝࠾ࡖࡒࠊࡱࡒࠉCdt1 ࢅඞ␷㝎ཡࡊࡒ༵᢫ ฝᾦ୯࡞ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊ࡙ࡵ Mcm6 ࡡ⤎ྙࡢ☔ヾ࡚ࡀ࡝࠾ࡖࡒ ࡒࡴࠉࡆࡿࡼࡡ᩷∞ࢰࣤࣂࢠ㈻࡞ࡢ Mcm2-7 々ྙమࡡࢠ࣏ࣞࢲࣤ⤎ྙࢅㄇᑙࡌ ࡾὩᛮࡢ࡝࠷ࡵࡡ࡛⩻࠻ࡼࡿࡒ (Figure 4-2)ࠊࡆࡡ⤎ᯕࡢࠉFigure 3-1 C ࡚♟ ࡊࡒCdt1 ᩷∞ࢰࣤࣂࢠ㈻࠿ DNA 々⿿ࣚ࢕ࢬࣤࢪ໩Ὡᛮࢅᣚࡒ࡝࠷࡛࠷࠹⤎ᯕ ࡛ྙ⮬ࡌࡾࡵࡡ࡚࠵ࡖࡒࠊ 㻫㼌㼖㻐㻦㼇㼗㻔㼈㼑㼇㼒㻑㻃㻦㼇㼗㻔㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦㻦㼇㼗㻔㻃㼙㼄㼕㼌㼄㼑㼗㻰㼆㼐㻙㼐㼒㼆㼎㻐㼗㼕㼈㼄㼗㼈㼇㻦㼇㼗㻔㻐㼇㼈㼓㼏㼈㼗㼈㼇㼅㼘㼉㼉㼈㼕㻦㼇㼗㻔㻱㻰㻦㻫㼌㼖㻐㻦㼇㼗㻔㻃㼙㼄㼕㼌㼄㼑㼗㼅㼘㼉㼉㼈㼕㻦㼇㼗㻔㻱㻰㻦㻫㼌㼖㻐㻦㼇㼗㻔㻃㼙㼄㼕㼌㼄㼑㼗 +LJ XU H -LV ( IW ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ ࢠ࣏ࣞ ࢲࣤ⤎ྙ ࠊ ඞ␷๑⾉ΰࠉᢘ (IW ⾉ΰࢅ⏕࠷࡙ඞ␷㝎ཡࡊࡒ༵᢫ฝᾦ࡞ࠉ⤂⃨ᗐ Q2 ࡡ -LV(IW3ࠉ 2ࠉ( ࢅ῟ຊࡊࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1ࢅ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊ ฦᚃ ࡞ࢠ࣏ࣞࢲࣤ⏤ฦࢅ༟㞫ࡊࠉ⾪♟ࡊࡒࢰࣤࣂࢠ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙࢅࢗ࢘ࢪࢰࣤࣇࣞࢴࢹ

Ἢ࡞ࡻࡽよᯊࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ =HQRSXV(IW ᢘమࠉ(IW ᩷∞ࢰࣤࣂࢠ㈻ࡡྜྷᏽ࡞

(19)

➠୔⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻࠿ DNA 々⿿࡞ཀྵ࡯ࡌᙫ㡢

ࡈࡼ࡞ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻࠿ DNA 々⿿࡞ཀྵ࡯ࡌᙫ㡢࡞ࡗ࠷࡙᳠ゞࡊࡒࠊ

Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡞࠽ࡄࡾ DNA 々⿿Ὡᛮࢅῼᏽࡊࡒ (Figure 4-3)ࠊࡱࡒࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ⁈よࡊ࡙࠷ࡾ buffer ࡡᙫ㡢ࢅ⩻៎

ࡊࠉ⢥⿿ࡊࡒ㝷ࡡ㏩ᯊbuffer ࢅ buffer control ࡛ࡊ࡙ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻⏤

ฦ࡛ྜྷᵕ࡞ᕵ㔐ࡊ⏕࠷࡙࠷ࡾࠊ !"#$%&'()*#+ #,,&#-.$/).! 0 1+!234.5267&87&3 09:2;<_=>&4?@= %#/!($#%#'.&AB C6?&!D'.E#!+! <;F <G&%+'H 1+! 234.5 26 1+! 234.5 28 1+! 234.5 23 I(BB#$&)A'H G JG 5GG G 5HG 34.5&B$/,%#'.&)A')H&&K L8M C6?& !D'.E#!+! K& N& AB& )A'.$A*M GHJ GH5 +LJ XU H (IW ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ )3& 々⿿࡫ ࡡᙫ㡢

-LV(IW3ࠉ2ࠉ( ࢅ⾪♟ࡡ⤂⃨ᗐ࡞࡝ࡾࡻ࠹࡞῟ຊࡊࠉ_{5I&95 Ꮛᅹୖ࡚⢥Ꮔᰶ )3&⤂⃨}

ᗐ ᮇµ1ࢅ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO ࡛ࡊ࡙ྜྷᵕࡡᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ ฦᚃ )3& ྙᠺ㔖ࢅῼᏽࡊࠉ*' GXIIHU ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡞࠽ࡄࡾ )3& ྙᠺ㔖ࢅ ࡛ࡊ࡙ )3& 々⿿ὩᛮࢅẒ㍉ࡊࡒࠊ

(20)

ࡐࡡ⤎ᯕࠉbuffer control ࡚ࡢⱕᖱࡡ DNA 々⿿Ὡᛮࡡ఩ୖ࠿ぜࡼࡿࡒࠊࡆࡿ

ࡢ㏩ᯊ buffer ୯ࡡ㧏⃨ᗐࡡሲࡷ⏲㟻Ὡᛮ๠࠿༵᢫ฝᾦ࡞୙࠻ࡒᙫ㡢࡚࠵ࡾ࡛⩻

࠻ࡼࡿࡾࠊHis-Cdt1-NࠉHis-Cdt1-C ࢅຊ࠻ࡒ༵᢫ฝᾦ࡚ࡢࠉbuffer control ࡛

ྜྷ⛤ᗐࡡDNA 々⿿Ὡᛮ࠿ヾࡴࡼࡿࡒࠊࡊ࠾ࡊ࡝࠿ࡼࠉCdc6 ࡛ࡡ⤎ྙ⬗ࢅࡵࡗ

His-Cdt1-M ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡚ࡢࠉbuffer control ࡛Ẓ㍉ࡊ࡙ࠉDNA 々⿿Ὡ ᛮ࠿ⴥࡊࡂ఩ୖࡊࡒ (Figure 4-3)ࠊ ࡆࡡ⤎ᯕࡻࡽࠉCdc6 ࡛හᅄᛮ Cdt1 ࡡ⤎ྙࢅ῟ຊࡊࡒ His-Cdt1-M ࠿㜴ᐐࡊ ࡒࡒࡴ࡞ࠉࣚ࢕ࢬࣤࢪ໩࡞␏ᖏࢅࡀࡒࡊDNA 々⿿Ὡᛮ࠿఩ୖࡊࡒࡡ࡚ࡢ࡝࠷࠾ ࡛࠷࠹ྊ⬗ᛮ࠿⩻࠻ࡼࡿࡒࠊࡐࡆ࡚Cdt1 ᩷∞ࢰࣤࣂࢠ㈻῟ຊ࡞ࡻࡾ DNA 々⿿ 㜴ᐐ࠿ࠉࣚ࢕ࢬࣤࢪ໩㜴ᐐ࡞ࡻࡾࡵࡡ࡝ࡡ࠾ḗ⟿࡚ࡈࡼ࡝ࡾ᳠ゞࢅ⾔࠹ࡆ࡛࡛ ࡊࡒࠊ ➠ᄿ⟿ His-Cdt1-M ࡞ࡻࡾ DNA 々⿿ࡡ㜴ᐐ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒ༵᢫ฝᾦࡡࣚ࢕ࢬࣤࢪ໩Ὡᛮࡡῼᏽࢅ⾔ࡖࡒࠊ ࣚ࢕ࢬࣤࢪ໩Ὡᛮࡡῼᏽࡢࠉ┘Ⓩࡡ᮪௲ࡡ༵᢫ฝᾦ࡞⢥Ꮔᰶ DNA ࢅຊ࠻ࠉ20 ฦ㛣 23Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊ࡙ࣚ࢕ࢬࣤࢪ໩ࢅᏰ஡ࡈࡎࡒᚃࠉ32_P-dATPࠉ geminin ࢅຊ࠻ࡒ᩺ࡒ࡝༵᢫ฝᾦ୯࡞⛛ࡊࠉ᩺⏍㙈࡫ࡡ 32_{P ཱིࡽ㎲ࡲ㔖ࢅᣞᵾ} ࡛ࡊ࡙ࣚ࢕ࢬࣤࢪ໩Ὡᛮ࡛ࡌࡾ᪁Ἢ࡚࠵ࡾ (Figure 4-4 ᕞ)ࠊࡱࡒ Cdt1 ᩷∞ࢰ ࣤࣂࢠ㈻ࢅ⁈よࡊ࡙࠷ࡾ buffer ࡡᙫ㡢ࢅ⩻៎ࡊࠉ⢥⿿ࡊࡒ㝷ࡡ㏩ᯊ buffer ࢅ buffer control ࡛ࡊ࡙ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻࡛ྜྷᵕ࡞ᕵ㔐ࡊ⏕࠷࡙࠷ࡾࠊࡐࡡ ⤎ᯕࠉHis-Cdt1-NࠉHis-Cdt1-C ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡚ࡢࠉbuffer control ࡛ྜྷ

(21)

! "! #!! ! #$! %&' ()*+# (, %&' ()*+# (-%&' ()*+# () ./00123456$ )*+#30278916+34564$33: ;-< =,>3 '?6+@1'&' :3 A3 503 456+25B< 'C12936/4B1& 18831D+274+' E3%&' ()*+# (FG3-G3) B&416'&68 021'@31D+274+' :3E819&6&6 G3HI(JK_LM*>NL3< 21CB&47+&56 917'/21916+350 =,>3'?6+@1'&' K$! !$# +LJ XU H -LV ( IW ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ ࣚ࢕ࢬ ࣤࢪ໩Ὡ ᛮ࡫ࡡ ᙫ㡢 -LV(IW3ࠉ2ࠉ( ࢅ⾪♟ࡡ⤂⃨ᗐ࡞࡝ࡾࡻ࠹࡞῟ຊࡊࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1 ࢅ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO

࡛ࡊ࡙ྜྷᵕࡡᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ ฦᚃࠉ྘ཬᚺ⁈ᾦࢅࠉJHPLQLQࠉ_{5I&95 ࢅ῟ຊࡊ}

ࡒ᩺㩥࡝༵᢫ฝᾦ࡞῟ຊࡊ Υ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ)3& ྙᠺ㔖ࢅῼᏽࡊࠉ*' GXIIHU ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡞࠽ࡄࡾ )3& ྙᠺ㔖ࢅ ࡛ࡊ࡙ )3& 々⿿ὩᛮࢅẒ㍉ࡊࡒࠊ

ࡗࡁ࡞ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒ༵᢫ฝᾦ୯࡚ࠉࣚ࢕ࢬࣤࢪ໩࠿Ᏸ஡

ࡌࡾཬᚺ㛜ጙ20 ฦᚃࡡ Cdt1ࠉCdc6ࠉMcm4ࠉMcm6 ࡡࢠ࣏ࣞࢲࣤ⤎ྙࢅ☔ヾ

ࡊࡒ (Figure 4-5)ࠊẒ㍉ᑊ㇗࡛ࡊ࡙ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ⢥⿿ࡊࡒ㝷ࡡ㏩ᯊ

buffer ࢅ buffer control ࡛ࡊ࡙⏕࠷࡙࠷ࡾࠊ

ࡐࡡ⤎ᯕࠉHis-Cdt1-NࠉHis-Cdt1-M ࡡ࠷ࡍࡿࢅ῟ຊࡊ࡙ࡵࠉහᅄᛮ Cdt1 ࡡ

ࢠ࣏ࣞࢲࣤ⤎ྙ࡞ን໩ࡢ࡝࠾ࡖࡒࠊୌ᪁ࠉHis-Cdt1-C ῟ຊ᮪௲ୖ࡚ࡢࢠ࣏ࣞࢲ

ࣤ⏤ฦ࡞࠽ࡄࡾ Cdt1 ࡡ΅ᑛ࠿ぜࡼࡿࡒࠊpull-down assay ࡞࠽࠷࡙ࡢࠉCdc6

(22)

࣏ࣞࢲࣤ⤎ྙ࡞࠽࠷࡙ࡢ❿ྙࡊ࡙࠷ࡾ࡛ࡢゕ࠻࡝࠾ࡖࡒ (Figure 4-5)ࠊࡱࡒࠉ ࣚ࢕ࢬࣤࢪ໩Ᏸ஡ࡡᣞᵾ࡛࡝ࡾ Mcm4ࠉMcm6 ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡗ࠷࡙ࡵࠉ His-Cdt1-NࠉHis-Cdt1-M ῟ຊ᮪௲ୖ࡚ࡡ㢟ⴥ࡝ን໩ࡢぜࡼࡿ࡝࠾ࡖࡒ࠿ࠉ His-Cdt1-C ࡚ࡢ 0.6 µM ࡚ⱕᖱ࡝࠿ࡼ⤎ྙ㔖࠿఩ୖࡊ࡙࠷ࡒ (Figure 4-5)ࠊࡒ ࡓࡊࠉࡆࡡHis-Cdt1-C ࡡຝᯕࡢ DNA 々⿿Ὡᛮ࡞ᙫ㡢ࢅ୙࠻ࡾ⛤ᗐ࡚ࡢ࡝࠷ࡵ ࡡ࡛⩻࠻ࡼࡿࡾࠊ 㻕㻖䉔㻕㻓㻃㼐㼌㼑㻑㼌㼐㼐㼘㼑㼒㼅㼏㼒㼗㼗㼌㼑㼊㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦㼈㼑㼇㼒㻑㻃㻦㼇㼗㻔㻱㻃㼒㼕㻃㻰㻃㼒㼕㻃㻦㼖㼓㼈㼕㼐㻃㼑㼘㼆㼏㼈㼌㼈㼊㼊㻃㼈㼛㼗㼕㼄㼆㼗㼖㻎㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻏㻃㻰㻏㻃㻦㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻓㻑㻙㻓㻑㻖㻓㻑㻔㻘㻓㻑㻙㻓㻑㻖㻓㻑㻔㻘㻓㻑㻙㻓㻑㻖㻓㻑㻔㻘㼅㼘㼉㼉㼈㼕㻃㼆㼒㼑㻑㻦㼇㼆㻙㻰㼆㼐㻗㻰㼆㼐㻙㻦㼇㼗㻔㻃㼉㼕㼄㼊㼐㼈㼑㼗㼆㼒㼑㼆㻑㻋㻃䃒㻰㻃㻌 +LJ XU H -LV ( IW ᩷∞ࢰ ࣤࣂࢠ ㈻࡞ࡻ ࡾහ ᅄᛮ (I W ࡡࢠ ࣏ࣞࢲ ࣤ⤎ྙ ࡫ࡡᙫ 㡢 -LV(IW3ࠉ2ࠉ( ࢅ⾪♟ࡡ⤂⃨ᗐ࡞࡝ࡾࡻ࠹࡞῟ຊࡊࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1ࢅ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO ࡛ ࡊ࡙ྜྷᵕࡡᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ ฦᚃ࡞ࢠ࣏ࣞࢲࣤ⏤ฦࢅ༟㞫ࡊࠉ⾪♟ࡊࡒࢰࣤࣂࢠ ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙࢅࢗ࢘ࢪࢰࣤࣇࣞࢴࢹἪ࡞ࡻࡽよᯊࡊࡒࠊ

(23)

Mcm2-7 々ྙమ࠿ࢠ࣏ࣞࢲࣤ࡞ᑙථࡈࡿ࡙࠷ࡾ࡛࠷࠹ࡆ࡛ࡢࠉࣚ࢕ࢬࣤࢪ໩ ࠿Ᏸ஡ࡊ࡙࠷ࡾࡆ࡛ࢅ♟ࡊ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡾࠊࡗࡱࡽࠉ๑㏑ࡊࡒࣚ࢕ࢬࣤࢪ ໩Ὡᛮࡡῼᏽ⤎ᯕ࡛□┢࠿⏍ࡋࡾࠊ௑ᅂࡡࣚ࢕ࢬࣤࢪ໩ὩᛮῼᏽἪࡢ᳠ゞᑊ㇗ ࡛࡝ࡾ᧧ష࠿ࣚ࢕ࢬࣤࢪ໩Ὡᛮ࡞ࡡࡲᙫ㡢ࢅ୙࠻ࡾ࡛࠷࠹ࡆ࡛࠿๑ᥞ࡚࠵ࡾࠊ ࡌ࡝ࢂࡔࠉHis-Cdt1-M ࠿ࣚ࢕ࢬࣤࢪ໩࡞ࡢᙫ㡢ࡢ୙࠻ࡍࠉࡐࡡᚃࡡ DNA 々⿿ 㛭㏻࢕࣊ࣤࢹ࡞ᙫ㡢ࢅ୙࠻࡙࠷ࡾ࡛ࡌࡿࡣࠉࣚ࢕ࢬࣤࢪ໩Ὡᛮࢅᢒโࡌࡾࡆ࡛ ࡝ࡂῼᏽ⤎ᯕࢅ఩ୖࡈࡎࡾࠊࡊࡒ࠿ࡖ࡙ࠉHis-Cdt1-M ࡡ༵᢫ฝᾦ࡫ࡡ῟ຊࡢࣚ ࢕ࢬࣤࢪ໩࡞ࡢᙫ㡢ࢅ୙࠻ࡍࠉDNA 々⿿ࡡࡐࡿ௧ᚃࡡṹ㝭ࢅᢒโࡌࡾ࡛⩻࠻ࡼ ࡿࡒࠊ ➠஫⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡ DNA 々⿿㛜ጙࠉఘ㛏ཬᚺ࡫ࡡᙫ㡢 Cdt1-M ࡡ῟ຊ࡞ࡻࡽࠉMcm2-7 々ྙమࡡࢠ࣏ࣞࢲࣤ࡫ࡡᑙථࢅᢒโ࡚ࡀ࡝࠾ ࡖࡒࡒࡴࠉFigure 4-3 ࡚ぜࡼࡿࡒ DNA 々⿿Ὡᛮࡡ఩ୖࡢࠉࣚ࢕ࢬࣤࢪ໩௧㜾ࡡ ṹ㝭࡫ࡡᙫ㡢࡚࠵ࡾࡆ࡛࠿⩻࠻ࡼࡿࡒࠊࡆࡿࡱ࡚Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅཬᚺ㛜 ጙ᫤࡞῟ຊࡊ࡙࠷ࡒ࠿ࠉࣚ࢕ࢬࣤࢪ໩࠿Ᏸ஡ࡌࡾ20 ฦᚃ࡞῟ຊࡊࠉࣚ࢕ࢬࣤࢪ ໩௧㜾࡞࠽࠷࡙ࡵDNA 々⿿ࢅ㜴ᐐࡊᚋࡾ࠾᳠ゞࡌࡾࡆ࡛࡛ࡊࡒࠊ ཬᚺ㛜ጙ᫤࠾ࡼCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒࡵࡡࠉཬᚺ㛜ጙࡻࡽ 20 ฦᚃ ࡞Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊࡒࡵࡡࡡ㸧⛸㢦࡞ࡗ࠷࡙ࠉཬᚺ㛜ጙ࠾ࡼ 90 ฦ

㛣ࡡDNA ྙᠺ㔖ࢅῼᏽࡊࡒ (Figure 4-6 A ᕞ)ࠊཬᚺ㛜ጙ 20 ฦᚃ࡞ His-Cdt1-Nࠉ

His-Cdt1-C ࢅ῟ຊࡊࡒ㝷࡞ࡢࠉ㛜ጙ᫤࡞῟ຊࡊࡒ㝷࡛Ẓ㍉ࡊ࡙ࠉⱕᖱࡡ々⿿Ὡ

ᛮࡡቌຊലྡྷࡢ࠵ࡖࡒࡵࡡࡡࠉ῟ຊ࡞ࡻࡾDNA 々⿿Ὡᛮࡡኬࡀ࡝ን໩ࡢࡷࡢࡽ

(24)

ࡒሔྙࡢࠉཬᚺ㛜ጙ᫤࡞῟ຊࡊࡒሔྙ࡛ྜྷᵕ࡞ DNA 々⿿Ὡᛮ࠿ⴥࡊࡂ఩ୖࡊࠉ ࢤࣤࢹ࣭࡛ࣞࣜẒ㍉ࡊ࡙⣑32%ࡡὩᛮࡊ࠾ヾࡴࡼࡿ࡝࠾ࡖࡒ (Figure 4-6 A)ࠊ ! "! #!! #"! $!! !%&'() $!%&'() *'+ ,-./# ,0 *'+ ,-./# ,1 *'+ ./# ,-233%24/567/+ 8%+925&%(:7;2' ;'72(+'(3 <52+=%24/567/+ >%8 32&'('( ?%@ A,B$_CD.EFC%G &26+:52&2(/%H< I0E%+J(/=2+'+ K:<<25 -./# ,0?%1?%--./# ,0?%1?%- K:<<25 $B L $!%&'() $B L M!%&'() I0E% +J(/=2+'+% >% N% H<% 7H(/5H;G 233%24/567/+ 8%+925&%(:7;2' ;'72(+'(3 <52+=%24/567/+ >%8 32&'('( ?% @A,B$_CD.EFC%G &26+:52&2(/%H< I0E%+J(/=2+'+ -./# ,1%H5%K:<<25 $B L $!%&'() $B L M!%&'() ! !)# #)! ! "! #!! K:<<25%7H() *'+,-./# ,1 -./#%<563&2(/%7H(7)%%> O1G I0E% +J(/=2+'+ >% N% H<% 7H(/5H;G !)" A B +LJ (I W ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ ࣚ࢕ࢬ ࣤࢪ໩ ௧㜾࡫ࡡ ᙫ㡢

&⤂⃨ᗐ µ2 ࡡ -LV(IW3ࠉ2ࠉ( ࡱࡒࡢ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ༵᢫ฝ ᾦ࡞῟ຊࡊࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1࡛࡛ࡵ࡞ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊ

ฦᚃࠉ྘ཬᚺ⁈ᾦ࡞ࠉJHPLQLQࠉ_{5I&95 ࢅຊ࠻ࡒ᩺㩥࡝༵᢫ฝᾦࢅ῟ຊࡊࠉ᭩ิ࡞ (IW ᩷}

∞ࢅ῟ຊࡊࡒモᩩ࡞ࡢ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅࠉ㏩ᯊ GXIIHU ࢅ῟ຊࡊࡒモᩩ ࡞ࡢྜྷ㔖ࡡ (IW ᩷∞ࢰࣤࣂࢠ㈻ࢅຊ࠻ࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ)3& ྙᠺ㔖 ࢅῼᏽࡊࡒࠊࢤࣤࢹ࣭࡛ࣞࣜࡊ࡙ ฦࠉ ฦ࡛ࡵ࡞ *'GXIIHU ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡞࠽ࡄ ࡾ )3& ྙᠺ㔖ࢅ ࡛ࡊ࡙ )3& 々⿿ὩᛮࢅẒ㍉ࡊࡒࠊ

'-LV(IW2 ࡡ )3& 々⿿Ὡᛮ࡫ࡡᙫ㡢 ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1ࢅ Υ࡚࢕ࣤ ࣭࢞ࣖ࣊ࢹࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO ࡛ࡊ࡙ྜྷᵕࡡ

ᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ ฦᚃࠉཬᚺ⁈ᾦ࡞ࠉJHPLQLQࠉ_{5I&95 ࢅຊ࠻ࡒ᩺㩥࡝༵᢫ฝᾦ}

(25)

ࡈࡼ࡞His-Cdt1-M ࢅ 20 ฦᚃ࡞῟ຊࡊࡒ㝷ࡡ DNA 々⿿Ὡᛮࡡ఩ୖ࡞ࡗ࠷࡙ His-Cdt1-M ࡡ⃨ᗐ࡞ᑊࡌࡾ౪Ꮛᛮࢅ᳠ゞࡊࡒ (Figure 4-6 B)ࠊࡐࡡ⤎ᯕࠉ His-Cdt1-M ⃨ᗐ౪ᏋⓏ࡞ DNA 々⿿Ὡᛮ࠿఩ୖࡊࠉཬᚺ㛜ጙ᫤࠾ࡼ῟ຊࡊࡒሔ ྙ (Figure 4-3 ཤ↯) ࡛ྜྷᵕࡡᣪິࢅ♟ࡌࡆ࡛࠿☔ヾࡈࡿࡒ (Figure 4-6 B)ࠊ ࡻࡖ࡙ His-Cdt1-M ࡡ῟ຊ࡞ࡻࡾ DNA 々⿿㜴ᐐࡢࠉࣚ࢕ࢬࣤࢪ໩ཬᚺ௧ᚃࡡ DNA 々⿿ࣆ࢚࣭ࢠࡡᙟᠺ࣬㐅⾔࡫ࡡᙫ㡢࡚࠵ࡾࡆ࡛࠿♟ြࡈࡿࡒࠊ Xenopus ༵᢫ฝᾦ୯࡚ࡡ DNA 々⿿࡞࠽࠷࡙ࠉཬᚺ㛜ጙ࠾ࡼ⣑ 20 ฦ࡚ Mcm2-7 々ྙమ࠿ࢠ࣏ࣞࢲࣤ࡞ᑙථࡈࡿࠉpre-RC ࠿ᙟᠺࡈࡿࡾࠊࡐࡡᚃ Mcm2-7 々ྙమ

࠿DDK (Dbf4-dependent kinase) ࡞ࡻࡖ࡙ࣛࣤ㓗໩ಞ㣥ࢅུࡄ࡙ pre-RC ࠿Ὡ

ᛮ໩ࡈࡿࡾ (Lei et al., 1997; Masai et al., 2005; Pereverzeva et al., 2000; Sato

et al., 1997)ࠊMcm4 ࠿ࣛࣤ㓗໩ࡈࡿࡾ࡛ᅒࡡࡻ࠹࡞ Mcm4 ࠿ SDS-PAGE ୕࡚

ࢪ࣒࢓≟ࡡࣁࣤࢺࢅ♟ࡌࡆ࡛࠿▩ࡼࡿࡾ (Figure 4-7, buffer control, 40 min.)ࠊ

ࡐࡡᚃࠉࣛࣤ㓗໩ࡈࡿࡒMcm2-7 々ྙమ࡞ Cdc45ࠉSld5-Psf1, 2, 3 (GINS) 々

ྙమ࡝࡜࠿⤎ྙࡊ (Jares and Blow, 2000; Walter, 2000; Zou and Stillman,

2000)ࠉ々⿿㉫Ⅴࡡ஦㔔㙈 DNA ᕬࡀᡘࡊࡷ DNA polymerase ࡡᑙථ࠿㛜ጙࡈࡿ

ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾ(Mimura et al., 2000; Walter and Newport, 2000)ࠊ

ࡐࡆ࡚ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ༵᢫ฝᾦ࡞῟ຊࡊࡒ㝷ࡡࢰࣤࣂࢠ㈻ࡡᣪິࢅࠉ ⤊᫤Ⓩ࡞☔ヾࡌࡾࡆ࡛࡛ࡊࡒࠊ_{Cdt1-M ࡢ 0.6 µM ࡚ᙁ࠷ DNA 々⿿㜴ᐐຝᯕࢅ} ♟ࡌࡒࡴࠉ0.6 µM ࡡ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ༵᢫ฝᾦ࡞῟ຊࡊࠉ᭩ኬ 90 ฦࡱ࡚ ࡡࢰࣤࣂࢠ㈻ࡡᣪິࢅ᥀ࡖࡒࠊࡌࡾ࡛ࠉHis-Cdt1-M ࢅ༵᢫ฝᾦ࡞῟ຊࡊࡒ㝷࡞ ࡢࠉMcm4 ࡡࢠ࣏ࣞࢲࣤ࠾ࡼࡡよ㞫࠿㐔ࡿ࡙࠷ࡾࡆ࡛ࠉ40 ฦᚃࡡ DNA polymerase αࡡࢠ࣏ࣞࢲࣤ⤎ྙ࠿ᘽࡂ࡝ࡖ࡙࠷࡙ࠉࢠ࣏ࣞࢲࣤ࠾ࡼࡡよ㞫ࡵ㐔 ࡿ࡙࠷ࡾࡆ࡛࠿☔ヾࡈࡿࠉDNA 々⿿࠿㐔ࡿ࡙࠷ࡾᵕᏄࢅ♟ࡊࡒ (Figure 4-7)ࠊ

(26)

ࡱࡒ 40 ฦᚃࡡ Mcm4 ࡡࣛࣤ㓗໩ࡡ⛤ᗐ࠿ᑊ↯࡛Ẓ㍉ࡊ࡙ ࠉᘽࡂ࡝ࡖ࡙࠷ࡒ (Figure 4-7)ࠊࡈࡼ࡞ࠉୌᮇ㙈 DNA ⤎ྙࢰࣤࣂࢠ㈻࡚࠵ࡾ replication protein A (RPA) 々ྙమࡡୌฦᏄ࡚࠵ࡾ Rpa30 ࡡ⤎ྙ࠿ࠉHis-Cdt1-M ࢅ῟ຊࡊࡒ㝷࡞ᘽ ࡂ࡝ࡖ࡙࠷ࡾࡆ࡛࠿☔ヾࡈࡿࡒ(Figure 4-7)ࠊࡻࡖ࡙ࠉHis-Cdt1-M ࡡ῟ຊ࡞ࡻ ࡽࠉ々⿿ࣆ࢚࣭ࢠࡡᙟᠺ࠿㐔ࡿ࡙࠷ࡾࡆ࡛࠿♟ြࡈࡿࡒࠊ 㻦㼇㼗㻔㻱䚮㻰䚮㻦㻰㼆㼐㻗㻳㼒㼏䃇㻦㼇㼆㻙㻓㻕㻓㻗㻓㻙㻓㻜㻓㻓㻕㻓㻗㻓㻙㻓㻜㻓㻓㻕㻓㻗㻓㻙㻓㻜㻓㻓㻕㻓㻗㻓㻙㻓㻜㻓㻷㼌㼐㼈㻃㻋㼐㼌㼑㻑㻌㼅㼘㼉㼉㼈㼕㻃㼆㼒㼑㼗㼕㼒㼏㻫㼌㼖㻐㻦㼇㼗㻔㻐㻱㻫㼌㼖㻐㻦㼇㼗㻔㻐㻰㻫㼌㼖㻐㻦㼇㼗㻔㻐㻦㻵㼓㼄㻖㻓 +LJ XU H -L V (I W ᩷ ∞ࢰࣤ ࣂࢠ㈻ ῟ຊ࡞ ࡻ ࡾ )3& 々⿿㛭㏻ࢰࣤ ࣂࢠ㈻ ࡡࢠࣞ ࣏ ࢲࣤ⤎ྙ ࡡ⤊᫤ Ⓩን໩ (IW ᩷∞ࢰࣤࣂࢠ㈻ࢅ⤂⃨ᗐ µ2 ࡞࡝ࡾࡻ࠹࡞༵᢫ฝᾦ࡞῟ຊࡊࠉ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1ࢅ Υ࡚࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO ࡛ࡊ࡙ྜྷᵕࡡᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ ฦࠉ ฦࠉ ฦࠉ ฦࠉ ฦ࡞ ࡐࡿࡑࡿࢠ࣏ࣞࢲࣤ⏤ฦࢅ༟㞫ࡊࠉ⾪♟ࡊࡒࢰࣤࣂࢠ㈻ࡡࢠ࣏ࣞࢲࣤ⤎ྙࢅࢗ࢘ࢪࢰࣤࣇ

ࣞࢴࢹἪ࡞ࡻࡽ᳠ฝࡊࡒࠊ(IW ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV(IW ᢘమࠉ(IH ࡡྜྷᏽ࡞ࡢᢘ =HQRSXV

(IH ᢘమࠉ(IW ᩷∞ࢰࣤࣂࢠ㈻ࡡྜྷᏽ࡞ࡢᢘ +1&, ᢘమࠉ2HP ࡡྜྷᏽ࡞ࡢᢘ=HQRSXV2HP

(27)

➠ඵ⟿ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻࡞ࡻࡾ DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㜴ᐐ ㎾ᖳࠉᙔ◂✪ᐄ࡞࠽࠷࡙ࠉCdt1 ࠿ DNA 々⿿࡞࠽ࡄࡾ᩺⏍㙈ఘ㛏ཬᚺࡡ㝷࡞ࠉ 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞ᑊࡊ࡙ᢒโⓏ࡞഼ࡂࡆ࡛ࢅぜฝࡊ࡙࠽ࡽࠉࣚ࢕ࢬࣤࢪ໩ ᅄᏄCdt1 ࡡ᩺ࡒ࡝ᶭ⬗࠿よ᪺ࡈࡿࡗࡗ࠵ࡾࠊࡐࡆ࡚ࠉHis-Cdt1-M ࡡ༵᢫ฝᾦ ࡫ࡡ῟ຊ࡞ࡻࡾDNA 々⿿Ὡᛮ఩ୖ࠿ࠉDNA 々⿿ࣆ࢚࣭ࢠࡡ㜴ᐐ࡞ࡻࡾࡵࡡ࡚ ࠵ࡾࡡ࠾࡞ࡗ࠷࡙ㄢ࡬ࡒ (Figure 4-8)ࠊ

ࡱࡍ⢥ᏄᰶDNA ࢅ㧏⃨ᗐ aphidicolin ࡛ caffeine ࢅ῟ຊࡊࡒ༵᢫ฝᾦ୯࡚ 60

ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡌࡾࠊ㧏⃨ᗐ aphidicolin ࡡ῟ຊࡢ DNA polymerase ࡞ᑊ

ࡌࡾ㢟ⴥ࡝㜴ᐐ࡛࡛ࡵ࡞ࠉࢲ࢘ࢴࢠ࣎࢕ࣤࢹᶭᵋࡡὩᛮ໩࡞ᇱࡘࡂ々⿿㉫ⅤὩ ᛮ໩ࡡᢒโࢅᘤࡀ㉫ࡆࡌࠊࢲ࢘ࢴࢠ࣎࢕ࣤࢹᶭᵋࡡ㜴ᐐ๠࡚࠵ࡾ caffeine ࢅ῟ ຊࡌࡾࡆ࡛࡚ࠉࡆࡡ々⿿㉫ⅤὩᛮ໩ࡡᢒโࢅよ㝎ࡊࠉᚃࡡఘ㛏ṹ㝭࡚ೳḾࡊࡒ ࢠ࣏ࣞࢲࣤࢅㄢ⿿ࡊࡒ (APH-chromatin)ࠊCDK 㜴ᐐࢰࣤࣂࢠ㈻࡚࠵ࡾ p21 ࢅ ྱࡳ༵᢫ฝᾦ୯࡞ࡆࡡࢠ࣏ࣞࢲࣤࢅ⛛ࡊࠉDNA 々⿿Ὡᛮࢅῼᏽࡊࡒ (Figure 4-8 ᕞ)ࠊࡆࡡṹ㝭࡚ࡢࠉp21 ࡡ῟ຊ࡞ࡻࡽࡈࡼ࡝ࡾ々⿿㉫ⅤὩᛮ໩ࢅᢒโࡈࡿ ࡙࠷ࡾࡒࡴࠉࡌ࡚࡞Ὡᛮ໩ࡈࡿࡒ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞࠽ࡄࡾᙫ㡢ࡡࡲࢅῼࡾ ࡆ࡛࠿࡚ࡀࡾࠊ ࡆࡡ᮪௲ୖ࡞࠽࠷࡙ࠉ⤂⃨ᗐ_{0.2 µM ࡡ GST-Cdt1 ࢅ῟ຊࡊࡒ࡛ࡆࢀࠉ㢟ⴥ࡝}

DNA 々⿿Ὡᛮࡡ఩ୖ࠿ふᐳࡈࡿࡒ (Figure 4-8 A)ࠊḗ࡞ࠉCdt1 ᩷∞ࢰࣤࣂࢠ

㈻ࢅ῟ຊࡊࡒ㝷࡞ࠉCdt1 ࡛ྜྷᵕࡡຝᯕ࠿ᚋࡼࡿࡾ࠾࡞ࡗ࠷࡙᳠ゞࡊࡒ࡛ࡆࢀࠉ

⤂⃨ᗐ࠿1.2 µM ࡚ࡵDNA 々⿿Ὡᛮࡡ఩ୖࡢ☔ヾ࡚ࡀ࡝࠾ࡖࡒ (Figure 4-8 B)ࠊ

ࡆࡿࡻࡽࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡢ DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞ᙫ㡢ࢅ୙࠻࡝࠷

(28)

!"# #"! $! #!! #$! %&'()*+# (, %&' ()*+# (-%&'()*+# () ./00123456+257 8,93 ':6+;1'&' <=3 503 456+257> )*+#302?@A16+34564"3< B-> ! 456+257 CDE()*+# ! #!! 8,93 ':6+;1'&' <=3 503 456+257> 'F12A36/471& 1@@31G+2?4+' H3?F;&*&457&6 4?001&61 !"#$ %&'()*+,-021';31G+2?4+' <3H3FI#J3K L(MI_NO*9EN3> CDE()*+# %&' ()*+#302?@A16+ A1?'/21A16+350 8,93':6+;1'&' IMP Q!A&6" 4;25A?+&63&'57?+&56 9 R +LJ XU H (IW ᩷∞ ࢰࣤࣂ ࢠ㈻ࡡ )3& 々⿿ࣆ ࢚࣭ࢠ㐅⾔㜴ᐐ ⢥Ꮔᰶ )3&⤂⃨ᗐ ᮇµ1ࢅ FSKLILHROLQ⤂⃨ᗐµJPOࠉHFIIHLQH⤂⃨ᗐP2 ࢅྱࡳ༵᢫ฝᾦ୯࡞ຊ࠻ࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡐࡡᚃࠉࢠ࣏ࣞࢲࣤ⏤ฦࢅ

༟㞫ࡊࠉSࠉ_@α_{5BI&95 ࢅྱࡳ᩺㩥࡝༵᢫ฝᾦ࡞῟ຊࡊࠉ}_{&⤂⃨ᗐ µ2 ࡡ ,89(IWࠉ}

'⾪♟ࡡ⤂⃨ᗐࡡ (IW ᩷∞ࢰࣤࣂࢠ㈻ࢅຊ࠻ࠉΥ࡚ ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉ )3& ྙᠺ㔖ࢅῼᏽࡊࡒࠊࡱࡒ (IW ᩷∞ࢰࣤࣂࢠ㈻ࡡ㏩ᯊ GXIIHU ࢅ GXIIHUHRQWURO ࡛ࡊ࡙ ྜྷᵕࡡᕵ㔐᧧షࢅ⾔࠷⏕࠷ࡒࠊ*'GXIIHU ࢅ῟ຊࡊࡒ༵᢫ฝᾦ࡞࠽ࡄࡾ )3& ྙᠺ㔖ࢅ ࡛ࡊ࡙ )3& 々⿿ὩᛮࢅẒ㍉ࡊࡒࠊ

(29)

➠஫❮ ⩻ᐳ

㸦ࠉCdt1 ࡡࢰࣤࣂࢠ㈻㛣┞பష⏕

㓕⣪Ὡᛮ࠿ྜྷᏽࡈࡿ࡙࠷࡝࠷Cdt1 ࡢࠉ௙ࡡࣚ࢕ࢬࣤࢪ໩ཬᚺ㛭㏻ࢰࣤࣂࢠ㈻

࡛┞பష⏕ࡊࠉ㏻ິⓏ࡞ᶭ⬗ࡌࡾࡆ࡛࠿㔔さ࡚࠵ࡾ࡛⩻࠻ࡼࡿ࡙ࡀࡒࠊࡆࡿࡱ

࡚࡞ࠉgemininࠉ࠵ࡾ࠷ࡢ Mcm2-7 々ྙమ࡛ࡡ⤎ྙ㡷ᇡ࠿ Cdt1 ࡞࠽࠷࡙᪺ࡼ࠾

࡛ࡈࡿ࡙ࡀࡒ (Ferenbach et al., 2005; Yanagi et al., 2002)ࠊୌ᪁ࠉᙔ◂✪ᐄ࡞

࠽࠷࡙Cdt1 ࡛ Cdc6 ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞᪺☔࡝㡨ᗆ࠿Ꮛᅹࡌࡾࡆ࡛࠿᪺ࡼ࠾࡛

࡝ࡽ (Tsuyama et al., 2005)ࠉCdt1 ࡛ Cdc6 ࡛ࡡ㛣࡞ࡵరࡼ࠾ࡡ┞பష⏕࠿Ꮛ

ᅹࡊࠉ୦⩽ࡢ༝ㄢⓏ࡞ᶭ⬗ࡊ࡙࠷ࡾࡡ࡚ࡢ࡝࠷࠾࡛࠷࠹ྊ⬗ᛮ࠿⩻࠻ࡼࡿࡒࠊ

ࡆࡿࡱ࡚ࠉCdt1 ࡛ Cdc6 ࠿├᥃⤎ྙࡊ࡙࠷ࡾ࡛࠷࠹ሒ࿈ࡢࡈࡿ࡙࠷࡝࠾ࡖࡒ

࠿ࠉᮇ◂✪࡚ࡢࠉCdt1 ࡡ᩷∞ࢰࣤࣂࢠ㈻ࢅ⏕࠷ࡒ pull down assay ࡞ࡻࡽࠉCdt1

࡛ Cdc6 ࠿⤎ྙࡊࠉࡈࡼ࡞ࡐࡡ⤎ྙࡢ Cdt1 ࡡ୯ኳ㒂 (Cdt1-M; 243-447) ࢅ௒ ࡊ࡙ࡊ࡙࠷ࡾ࡛࠷࠹ࡆ࡛ࢅ᪺ࡼ࠾࡞ࡊࡒ (Figure 3-2)ࠊ ࡱࡒᮇ◂✪࡞࠽࠷࡙ࠉࢰࢡࡡ␏࡝ࡾCdt1 ࢅ⏕࠷ࡒ pull-down assay ࡞ࡻࡽࠉ Cdt1 ྜྷኃ࠿┞பష⏕ࡌࡾࡆ࡛ࢅ᪺ࡼ࠾࡞ࡊࡒ (Figure 3-5)ࠊࡈࡼ࡞ࠉCdt1 ᩷ ∞ࢰ ࣤ ࣂࢠ㈻ࢅ⏕ ࠷ࡾ ࡆ ࡛ ࡚ࠉࡐ ࡡ ┞பష ⏕ ࡢ Cdt1 ࡡ୯ኳ㒂 (Cdt1-M; 243-447) ࡡ㡷ᇡࢅ୯ᚨ࡞⾔ࢂࡿ࡙࠷ࡾࡆ࡛ࢅ♟ࡊࡒ (Figure 3-6)ࠊࡆࡿࡱ࡚ࠉ ⢥Ꮔᰶ DNA ࠿Ꮛᅹࡊ࡝࠷༵᢫ฝᾦ୯࡞࠽࠷࡙ࠉCdt1 ࠿࡜ࡡࡻ࠹࡝ᙟ࡚Ꮛᅹࡊ ࡙࠷ࡾ࠾࡞ࡗ࠷࡙ࡢ᪺ࡼ࠾࡛ࡈࡿ࡙࠷࡝࠾ࡖࡒࠊࡊ࠾ࡊ࡝࠿ࡼࠉᮇ◂✪ࡡ⤎ᯕ ࠾ࡼࠉࣚ࢕ࢬࣤࢪ໩ࡡ୯ᚨⓏᙲ๪ࢅᢰ࠹Cdt1 ࠿ࠉ༵᢫ฝᾦ୯࡞࠽࠷࡙ᏭᏽⓏ࡞ Ꮛᅹࡌࡾࡒࡴ࡞ࠉኣ㔖మࢅᙟᠺࡊ࡙࠷ࡾྊ⬗ᛮࢅ♟ࡊࡒࠊ

(30)

ᙔ◂✪ᐄ࡚ࡢࠉCdt1 ࡛ Cdc6 ࠿⤎ྙࡊࡒ≟ឺ࡚ࠉ⢥Ꮔᰶ DNA ࢅຊ࠻࡙࠷࡝ ࠷༵᢫ฝᾦ୯࡞ຊ࠻ࡾ࡛ࠉ⤎ྙ࠿よ㝎ࡈࡿࡾࡆ࡛ࢅぜฝࡊ࡙࠷ࡾࠊࡌ࡝ࢂࡔࠉ ୦⩽ࡡ⤎ྙࡢࠉ༵᢫ฝᾦ୯࡚ࡢరࡼ࠾ࡡᙟ࡚โᚒࡈࡿ࡙࠽ࡽࠉ⢥ᏄᰶDNA ῟ຊ ࡡᚃࠉࢠ࣏ࣞࢲࣤ୕࡚ࡡࡲ୦⩽࠿┞பష⏕ࡌࡾ࡛࠷࠹ࡆ࡛࠿⩻࠻ࡼࡿࡾࠊ ୦⩽ࡡ┞பష⏕࠿༵᢫ฝᾦ୯࡚โᚒࡈࡿ࡙࠷ࡾཋᅄ࡛ࡊ࡙ࡢࠉCdt1ࠉࡵࡊࡂ ࡢ Cdc6 ࠿࡮࠾ࡡࢰࣤࣂࢠ㈻࡛┞பష⏕ࡊ࡙࠷ࡾࡒࡴ࡛࠷࠹ࡆ࡛ࡵ⩻࠻ࡼࡿࡾࠊ M ᭿࡚ࡢ Cdt1 ࡢ㜴ᐐࢰࣤࣂࢠ㈻࡚࠵ࡾ geminin ࡛⤎ྙࡊ࡙࠽ࡽࠉࡐࡡᶭ⬗࠿ ᢒโࡈࡿ࡙࠷ࡾࠊanaphase-promoting complex (APC) ࢅ௒ࡊࡒࣗࣄ࢞ࢲࣤ໩

⤊㊪࡞ࡻࡾgeminin ࡡฦよ࠿ࠉM ᭿ࢅ⬲ฝࡊࡒࡡࡔ࡞ DNA 々⿿ࢅ㐅⾔ࡈࡎࡾ

ࡒࡴ࡞ᚪさ࡚࠵ࡾ (Hodgson et al., 2002; McGarry and Kirschner, 1998)ࠊᮇ

◂✪࡚⏕࠷࡙࠷ࡾ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ᢫ฝᾦࡢࠉgeminin ࠿ฦよࡈࡿࡒ࠵࡛ ࡡG1 ᭿ࡡࡵࡡ࡚࠵ࡾ࡛⩻࠻ࡼࡿࡾࡒࡴࠉCdt1 ࠿ geminin ࡛⤎ྙࡊ࡙࠷ࡾ࡛ࡢ ⩻࠻࠿ࡒ࠷ࠊࡐࡡࡒࡴࠉ᩺ࡒ࡝Cdt1 โᚒࢰࣤࣂࢠ㈻࠾ࠉࡵࡊࡂࡢ Figure3-5 ࡚ ♟ࡊࡒࡻ࠹࡞Cdt1 ྜྷኃࡡኣ㔖మᙟᠺ࡝࡜࠿ཋᅄ࡛ࡊ࡙⩻࠻ࡼࡿࡾࠊ ௑ᚃࡢࠉࡆࡿࡼࡡࢰࣤࣂࢠ㈻㛣┞பష⏕ࡡࡈࡼ࡝ࡾレ⣵࡝よᯊ࡛࡛ࡵ࡞ࠉ༵ ᢫ฝᾦ୯࡚ᐁ㝷࡞㉫ࡆࡽ࠹ࡾ┞பష⏕࡚࠵ࡾࡡ࠾᳠ゞࡌࡾᚪさ࠿࠵ࡾࠊ 㸧ࠉࢠ࣏ࣞࢲࣤ୕࡞࠽ࡄࡾ Cdt1-Cdc6 ┞பష⏕ࡡ㜴ᐐ ᮇ◂✪ࡢࠉCdt1 ࡛ Cdc6 ࡡ┞ப౪Ꮛᛮࢅ᪺ࡼ࠾࡛ࡌࡾࡆ࡛ࢅᙔิࡡ┘Ⓩ࡛ࡊ ࡙࠷ࡒࠊࡊ࠾ࡊ࡝࠿ࡼࠉHis-Cdt1-M ࡡ῟ຊ࡞ࡻࡖ࡙ࡵ୦⩽ࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ ᑊࡌࡾ❿ྙࡷࠉࣚ࢕ࢬࣤࢪ໩ཬᚺࡡ㜴ᐐࡢ☔ヾ࡚ࡀ࡝࠾ࡖࡒ (Figure 4-5)ࠊCdt1

(31)

࡞౪Ꮛࡌࡾ࠾ྫྷ࠾࡞ࡗ࠷࡙ࡢ☔ヾ࡚ࡀ࡙࠷࡝࠷ࠊࡊࡒ࠿ࡖ࡙ࠉ᩷∞໩ࡈࡿ࡙࠷ ࡾࡒࡴ࡞ORC ౪Ꮛᛮ࠿኶ࢂࡿࠉ々⿿㉫Ⅴ㎾എ࡚ࡢ࡝ࡂࠉࢠ࣏ࣞࢲࣤ࡞ࣚࣤࢱ࣑ ࡞⤎ྙࡊ࡙ࡊࡱࡖࡒࡒࡴ࡞ࠉහᅄᛮCdt1 ࡛ࡡ❿ྙᛮ࠿☔ヾ࡚ࡀ࡝࠾ࡖࡒ࡛࠷࠹ ࡆ࡛ࡵ⩻࠻ࡼࡿࡾࠊ௑ᚃࡢࠉCdc6 ࡛ࡡ┞பష⏕㡷ᇡࢅࡈࡼ࡞レ⣵࡞≁ᏽࡌࡾࡆ ࡛ࡷࠉORC ࡛ࡡ⤎ྙ㡷ᇡࢅ᪺ࡼ࠾࡛ࡌࡾᚪさ࠿࠵ࡾ࡛⩻࠻࡙࠷ࡾࠊ 㸨ࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡ DNA 々⿿࡫ࡡᙫ㡢 ᮇ◂✪࡚ࡢࠉࡐࡿࡑࡿ࠿≁ᏽࡡᶭ⬗㡷ᇡ࡛ࡊ࡙ヾࡴࡼࡿ࡙࠷ࡾ Cdt1-Nࠉ Cdt1-MࠉCdt1-C ࡡ㡷ᇡࡡ⤄ᥦ࠻ࢰࣤࣂࢠ㈻࠿ DNA 々⿿࡞࡜ࡡࡻ࠹࡝ᙫ㡢ࢅཀྵ ࡯ࡌ࠾࡞ࡗ࠷࡙᳠ゞࡊࡒࠊCdt1-N ࡡ㡷ᇡࡢ Cdt1 ࡡฦよโᚒ࡞㛭ࢂࡖ࡙࠽ࡽࠉ His-Cdt1-N ࢅ㧏⃨ᗐ῟ຊࡊࡒ༵᢫ฝᾦ୯࡚ࡢࠉCdt1 ࡡฦよ࠿ᢒโࡈࡿࡒ (Figure 4-1)ࠊ᭩㎾ࠉࣃࢹ⣵⬂࡞࠽࠷࡙ Cdt1-N ࢅ㐛๨࡞Ⓠ⌟ࡈࡎࡒ㝷࡞ࠉ්々 ⿿ࢅㄇᑙࡌࡾ࡛࠷࠹ሒ࿈࠿࡝ࡈࡿࡒ (Teer and Dutta, 2008)ࠊࡊ࠾ࡊ࡝࠿ࡼࠉ

ᮇ◂✪࡞࠽ࡄࡾᐁ㥺᮪௲࡚ࡢࠉDNA 々⿿Ὡᛮ࡞ࡢᙫ㡢࠿࡮࡛ࢆ࡜ぜࡼࡿࡍࠉ්

々⿿࠿ㄇᑙࡈࡿࡾࡆ࡛ࡵ࡝࠾ࡖࡒ (Figure 4-2)ࠊCdt1-C ࡢࠉMcm2-7 々ྙమࡡ Mcm6 ࡛⤎ྙࡌࡾࡆ࡛࠿ሒ࿈ࡈࡿ࡙࠷ࡾ (Ferenbach et al., 2005)ࠊHis-Cdt1-C

ࢅ㐛๨࡞῟ຊࡊࡒ㝷࡞ࡢࠉDNA 々⿿Ὡᛮ࡞ࡢᙫ㡢ࡢぜࡼࡿ࡝࠾ࡖࡒ࠿ࠉMcm2-7 々ྙమࡡࢠ࣏ࣞࢲࣤ⤎ྙ࠿఩ୖࡊࡒ (Figure 4-3ࠉ4-5)ࠊࡆࡿࡢහᅹᛮࡡ Cdt1 ࡛ Mcm2-7 々ྙమ࡛ࡡ┞பష⏕࠿ Cdt1-C ࡞ࡻࡖ࡙❿ྙⓏ࡞㜴ᐐࡈࡿࡾࡒࡴ࡚ ࠵ࡾࡡ࠾ࡵࡊࡿ࡝࠷ࠊCdt1-M ࡢࠉᮇ◂✪࡚᪺ࡼ࠾࡛࡝ࡖࡒ࡛࠽ࡽࠉCdc6 ࡛ࡡ ⤎ྙ⬗ࢅࡵࡗࠊࡊ࠾ࡊ࡝࠿ࡼࠉHis-Cdt1-M ࢅ῟ຊࡊࡒ༵᢫ฝᾦࡢ DNA 々⿿Ὡ ᛮ࠿ኬࡀࡂ఩ୖࡊࡒ࡞ࡵ࠾࠾ࢂࡼࡍࠉㄢ࡬ࡒ㝀ࡽࡡࣚ࢕ࢬࣤࢪ໩㛭㏻ࢰࣤࣂࢠ ㈻࡞ࡗ࠷࡙ࠉࡐࡿࡼࡡࢠ࣏ࣞࢲࣤ⤎ྙ࡞ࡢᙫ㡢ࡢ࡝࠾ࡖࡒ (Figure 4-3ࠉ4-5)ࠊ

(32)

㎾ᖳࠉᙔ◂✪ᐄ࡞ࡻࡽࠉCdt1 ࠿ DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞ᢒโⓏ࡞഼ࡂࡆ ࡛ࠉࡱࡒ geminin ࡛ᣍᢘࡊ࡙々⿿୯ࡡ්々⿿ࢅᢒโࡊ࡙࠷ࡾ࡛࠷࠹ Cdt1 ࡡ᩺ ࡒ࡝ᶭ⬗ࢅぜฝࡊ࡙࠷ࡾ (⌟ᅹᢖ✇୯)ࠊࡐࡆ࡚ࠉCdt1-M ࡞ࡻࡾ DNA 々⿿ࡡ఩ ୖ࠿ Cdt1 ࡛ྜྷᵕࡡຝᯕ࡞ࡻࡽࡵࡒࡼࡈࡿࡾࡡ࠾࡞ࡗ࠷࡙᳠ゞࡊࡒࠊࡐࡡ⤎ᯕࠉ Cdt1-M ࡡ῟ຊࡢ DNA 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔࡞ࡢᙫ㡢ࡢ୙࠻࡝࠷ࡆ࡛࠿♟ြࡈࡿ ࡒ(Figure 4-8)ࠊ Cdt1 ࡡ DNA 々⿿ࣆ࢚࣭ࢠ㐅⾔ࡡᢒโࡢ々⿿ࣆ࢚࣭ࢠ୯ࡡరࡼ࠾ࡡࢰࣤࣂࢠ ㈻࡛ࡡ┞பష⏕࠿ᙫ㡢ࡊ࡙࠷ࡾ࡛஢᝷ࡈࡿࡾ࠿ࠉレ⣵࡝࣒࢜ࢼࢫ࣑࡞ࡗ࠷࡙ࡢ ୘᪺࡝Ⅴ࠿ኣ࠷ࠊ⌟ᅹࠉDNA 々⿿ࣆ࢚࣭ࢠ୕࡞Ꮛᅹࡌࡾࢰࣤࣂࢠ㈻ࡡ୯࡚ࠉCdt1 ࡛ࡡ┞பష⏕࠿▩ࡼࡿ࡙࠷ࡾࡡࡢ Mcm2-7 々ྙమ࡚࠵ࡾࠊࢨࣘࢗࢩࣘࢗࣁ࢙ࢅ ⏕࠷ࡒ◂✪࡚ࠉDNA 々⿿ࣆ࢚࣭ࢠࡡ࡝࠾࡚ Mcm2-7 々ྙమࡢࠉGINS ࡛ Cdc45 ࡛々ྙమࢅᙟᠺࡊ࡙ࣉ࣭ࣛ࢜ࢭ࡛ᶭ⬗ࡊ࡙࠷ࡾ࡛࠷࠹ࡆ࡛࠿ሒ࿈ࡈࡿ࡙࠷ࡾ (Gambus et al., 2006; Moyer et al., 2006)ࠊࡆࡡࡒࡴࠉMcm6 ࡛ࡡ⤎ྙ⬗ࢅᣚࡗ Cdt1-C ࡡ῟ຊ࡞ࡻࡽྜྷᵕࡡ⤎ᯕ࠿ᚋࡼࡿࡾࡡ࡚ࡢ࡝࠷࠾࡛᭿ᙽࡊࡒ࠿ࠉ≁࡞ᙫ 㡢ࡢぜࡼࡿ࡝࠾ࡖࡒࠊᮇ◂✪࡚౐⏕ࡊࡒࡻ࠹࡝᩷∞໩ࡊࡒ Cdt1 ࡚ࡢࠉCdt1 ⮤ మࡡᶭ⬗࠿࡮࡛ࢆ࡜኶ࢂࡿ࡙ࡊࡱࡖ࡙࠷ࡾࡒࡴ࡞ࠉྜྷᵕࡡ⌟㇗࠿ぜࡼࡿ࡝࠾ࡖ ࡒ࡛࠷࠹ྊ⬗ᛮࡵ࠵ࡾࠊࡐࡡࡒࡴࠉࡆࡡ⌟㇗࡞㛭ࡊ࡙ࡢCdt1 ࡡᶭ⬗ࡡḖ᥾ࢅ᭩ ᑚ㝀࡞ࡊࡒCdt1 mutant ࡚᳠ゞࡌࡾᚪさ࠿࠵ࡾ࡛⩻࠻࡙࠷ࡾࠊ Cdt1-M ࡡ῟ຊ࡞ࡻࡽࠉMcm4 ࡡࣛࣤ㓗໩࠿΅ᘽࡊ࡙࠽ࡽࠉࡱࡒ DNA polymerase Șࡡࢠ࣏ࣞࢲࣤ࡫ࡡ⤎ྙ࠿΅ᑛࡊ࡙࠷ࡒࡆ࡛࠾ࡼࠉDNA 々⿿㛜ጙ 々ྙమᙟᠺࡷࡐࡡὩᛮ໩ࡡ㐔ࡿ࠿ DNA 々⿿Ὡᛮࡡ఩ୖࡡཋᅄ࡚࠵ࡾࡆ࡛࠿♟ ြࡈࡿࡒ (Figure 4-7)ࠊࢠ࣏ࣞࢲࣤ୕࡞⤎ྙࡊࡒ Cdt1-M ࠿ࠉᵋ㏸Ⓩ࡞ Mcm4 ࡡࣛࣤ㓗໩ࢅ㜴ᐐࡊࡒࡒࡴ࡞ࠉࡆࡡࡻ࠹࡝⤎ᯕ࠿ᚋࡼࡿࡒࡡ࡚ࡢ࡝࠷࠾࡛⩻࠻

(33)

ぜฝࡊ࡙࠽ࡽࠉࡆࡿࡼࡢࣚ࢕ࢬࣤࢪ໩㛭㏻ࢰࣤࣂࢠ㈻ࡡโᚒ࡛ Mcm2-7 ࡡὩᛮ ໩࠿ᐠ᥃࡞㏻ິࡊ࡙࠷ࡾྊ⬗ᛮࢅ♟ࡊ࡙࠷ࡾࡵࡡ࡛⩻࠻࡙࠷ࡾࠊࡌ࡝ࢂࡔࠉࡆ ࡡࡻ࠹࡝⌟㇗ࡵཚᐠ࡞โᚒࡈࡿࡒࣚ࢕ࢬࣤࢪ໩ᶭᵋࡡୌ㒂࡚࠵ࡾ࡛ゕ࠻ࡾ࠾ࡵ ࡊࡿ࡝࠷ࠊࡐࡡណ࿝࡚ࡢࠉCdc6 ⤎ྙ㡷ᇡࢅᣚࡗ Cdt1-M ࡡ῟ຊ࠿ Cdc6 ࡡ㐛๨ ῟ຊ࡛ྜྷᵕࡡຝᯕࢅࡵࡒࡼࡌࠉ࡛࠷࠹ࡆ࡛ࡢ⮾࿝῕࠷ࠊ ࡊ࠾ࡊ࡝࠿ࡼࠉ々⿿ࣆ࢚࣭ࢠࡡ㐔ࡿࡡ⛤ᗐࡢ㢟ⴥ࡝ࡵࡡ࡚࠵ࡾ࡛ࡢゕ࠷㞬ࡂࠉ DNA 々⿿Ὡᛮࢅⴥࡊࡂ఩ୖࡈࡎࡾ။ୌࡡཋᅄ࡚࠵ࡾ࡛࠷࠹࡞ࡢࠉⱕᖱࡡ␪ၡ࠿ ṟࡾࠊ᚜ࠍࡒࡾᵕࠍ࡝さᅄ࠿㔔࡝ࡽࠉDNA 々⿿Ὡᛮࡡኬࡀ࡝఩ୖ࡞⦽࠿ࡖࡒ࡛ ⩻࠻ࡾࡡ࠿ጂᙔࡓ࡛ᛦࢂࡿࡾࠊ ௑ᚃࡡᒈ᭻ Cdt1 ࡢ DNA 々⿿㛜ጙโᚒᶭᵋ࡞࠽࠷࡙୯ᚨⓏ࡝ᙲ๪ࢅᢰ࠹ࢰࣤࣂࢠ㈻࡚࠵ ࡾ࠿ࠉࡐࡡᶭ⬗࡞ࡢ୘᪺࡝Ⅴ࠿ኣ࠷ࠊᶭ⬗ࢺ࣒࢕ࣤ࡞㛭ࡌࡾよᯊࡵࡐࡿ࡮࡜⾔ ࢂࡿ࡙࠷࡝࠷ࠊࡐࡡཋᅄࡡୌࡗ࡛ࡊ࡙ࠉኣࡂࡡን␏ᆵCdt1 ⤄ᥦ࠻ࢰࣤࣂࢠ㈻ࡢ ⁈よࡊ࡞ࡂࡂࠉ⢥⿿࠿㟸ᖏ࡞ᅏ㞬࡚࠵ࡾࡆ࡛࠿ᣪࡅࡼࡿࡾࠊ≁࡞ Cdt1-M 㡷ᇡ ࡢࡀࢂࡴ࡙୘⁈ᛮࡡ㧏࠷㡷ᇡ࡚࠵ࡾࠊࡱࡒࠉᮇ◂✪࡚౐⏕ࡊࡒ᩷∞໩Cdt1 ࡚ࡢ Ḗ᥾࠿ኬࡀࡌࡁࡾࡒࡴࠉᵋ㏸࡛≁ᏽࡡ⏍໩ᏕⓏὩᛮࡡ㛭㏻࡞ࡗ࠷࡙ࡡレ⣵࡝よ ᯊ࡞ࡢ᭯ຝ࡚࠵ࡖ࡙ࡵࠉ࠷ࡂࡗ࠾ࡡ⣪ཬᚺ࠿㏻ᦘࡊ࡙Ⓠ⌟ࡌࡾࡻ࠹࡝ᶭ⬗ࡡよ ᯊ࡞ࡢ᭯ຝ࡚ࡢ࡝࠷࡛ᛦࢂࡿࡾࠊࡆࡡࡻ࠹࡝ሔྙ࡞ࡢࠉ㒂ฦⓏ࡞Ḗ኶ࡊࡒ mutant ࢅ⏕࠷ࡾࡆ࡛࠿ᚪさ࡝ࡡ࠾ࡵࡊࡿ࡝࠷ࠊ ᮇ◂✪࡚ࡢࠉCdt1 ᩷∞ࢰࣤࣂࢠ㈻ࢅ⏕࠷࡙ࠉ᩺ࡒ࡞ Cdc6 ࡡ⤎ྙ㡷ᇡࠉCdt1 ྜྷኃࡡ⤎ྙ㡷ᇡࢅྜྷᏽࡊࡒࠊࡱࡒࠉࣚ࢕ࢬࣤࢪ໩࡛ Mcm2-7 々ྙమࡡὩᛮ໩࠿ ᐠ᥃࡞㏻ິࡊ࡙࠷ࡾྊ⬗ᛮࢅ♟ြࡊࡒࠊᮇ◂✪ࡡよᯊࢅ㊂࠿࠾ࡽ࡞ࠉࡈࡼ࡝ࡾ

(34)

Cdt1 ࡡᶭ⬗ࡡよ᪺࠿⾔ࢂࡿࡾࡆ࡛࠿᭻ࡱࡿࡾࠊ ➠ඵ❮ ᐁ㥺ᮞᩩཀྵࡦᐁ㥺᪁Ἢ ௧ୖ࡞♟ࡌᮞᩩཀྵࡦモⷾ࡞㛭ࡊ࡙ࠉ≁࡞᪺オࡊ࡙࠷࡝࠷㝀ࡽSigma-Aldrichࠉ NacalaiࠉWako ࡡࡵࡡࢅ౐⏕ࡊࡒࠊ ➠ୌ⟿ ࢓ࣆࣛ࢜ࢵ࣒࢙༵࢝ࣜ᢫ฝᾦ (ULSS)ࡡㄢᩒ 1.1 ᮞᩩ䛮モⷾ 1) ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ 㞜 (࢜ࢹ࣭S ࢜࢝ࢠ) 2) Chorionic gonadotropin (⫶┑ᛮᛮ⭚ๆ⃥࣓࣌ࣜࣤ) (᪝ᮇධⷾᕝᴏ) 3) modified high salt barth (MHSB)

110 mM NaClࠉ15 mM Trisࠉ2 mM KClࠉ1 mM MgCl2

HCl ࡚ pH7.6 ࡞ㄢᩒࡊࡒࠊ 4) cystein buffer

2.9% cystein-HCl-H20ࠉ1 mM EGTA (ethyleneglycol-bis-(2-amino

ethylether)-N,N,N’,N’-tetraacetic acid) (Fkluka) NaOH ࡚ pH7.5 ࡞ㄢᩒࡊࡒࠊ

5) high salt barth (HSB)

110mM NaClࠉ15 mM Trisࠉ2 mM KClࠉ1 mM MgSO4ࠉ2 mM

NaHCO3ࠉ

0.5 mM Na2HPO4ࠉ2 mM EGTA (Fluka)

(35)

6) unactivated extraction buffer (UEB) 50 mM KClࠉ50 mM HEPES-KOH (pH7.6)ࠉ5 mM MgCl2ࠉ5 mM EGTA (Fluka) ౐⏕├๑࡞2-mercaptoethanol ࢅ῟ຊࡊࡒ (⤂⃨ᗐ 2 mM)ࠊ 7) cytochalasin B ⁈ᾦ cytochalasin B ࢅ⤂⃨ᗐ 10 mg/ml ࡞࡝ࡾࡻ࠹࡞ DMSO ࡞⁈よࡊ -20Υ࡚ಕᏋࡊࡒࠊ

8) extract dilution buffer with sucrose (EDB-S)

50 mM KCl ࠉ 50 mM HEPES-KOH (pH7.6) ࠉ 2 mM DTT

(dithiothreitol)ࠉ0.4 mM MgCl2ࠉ0.4 mM EGTAࠉ10% sucroseࠉ

10 µg/ml pepstatin A (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ 10 µg/ml leupeptin (࣋ࣈࢲࢺ◂✪ᡜ)

1.2 ᪁Ἢ

࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ㞜ࡡ⏐༵ࢅㄇᑙࡈࡎࡾࡒࡴࠉ༵᢫ฝᾦࢅㄢ⿿ࡌࡾ๑᪝ࡡ

ኟ᪁࡞ chorionic gonadotropin 200 U/༆ࢅ⓮ୖᢖ୙ࡊࠉ2 ᅂ┘ࡡᢖ୙௧㜾ࡢ

MHSB 2 l/༆࡚㣣⫩ࡊࡒࠊỀᵬࡡῺᗐࡢ 18Υ࠾ࡼ༵ࢅᅂ཭ࡌࡾ 2 ᫤㛣๑࡞ 22Υ ࡞࡝ࡾࡻ࠹࡞スᏽࡊࡒࠊ

ᙔ᪝ࡢࡱࡍ༵ࢅᅂ཭ࡊࠉ㈻ࡡᝇ࠷༵ࢅ㝎࠷ࡒࠊMHSB ࢅ࡚ࡀࡾ㝀ࡽ㝎ࡀࠉ cystein buffer ࢅຊ࠻࡙㟴⨠ࡌࡾࡆ࡛࡚ᐨኮ㈻ࢅ㝎࠷ࡒࠊ3 ᅂ࡮࡜ cystein buffer

࡚ὑࡖࡒᚃࠉᐄῺࡡ HSBࠉ4Υ࡞෫ࡷࡊࡒ UEB ࡚ࡐࡿࡑࡿ 3 ᅂࡍࡗὑࡖࡒࠊ

UEB ࢅ࡚ࡀࡾ㝀ࡽ㝎ࡀࠉFalcon2059 ࡞⛛ࡊ࡙㐪ᚨฦ㞫 (2,220 rpmࠉ4Υࠉ2 min) ࡊࠉ୕ΰ࡛ࠉ༵ࡡᒒࡡ⾪㟻࡞㞗ࡱࡖࡒ㈻ࡡᝇ࠷༵ࢅ㝎࠷ࡒࠊ්ࡦ㐪ᚨฦ㞫 (8,960

(36)

rpmࠉ4Υࠉ12 min) ࡊ୕㒂ࡡ㯜Ⰵ࠷ࢠ࣭࣑ࣛ≟ࡡᒒࢅཱིࡽ㝎࠷ࡒᚃࠉ୯ኳ㒂ࡡ ࢠ࣭࣑ࣛⰅ㹳㯦》ⰅࡡᒒࡓࡄࢅFalcon2059 ࡞ᅂ཭ࡊࡒࠊࡆࡿ࡞ 1/1000 ᐖ㔖ࡡ cytochalasin B ⁈ᾦ࡛ 15%ᐖ㔖ࡡ EDB-S ࢅຊ࠻࡙ࡻࡂΊ࿰ࡊࡒࠊࡆࡿࢅ㐪ᚨฦ 㞫 (10,000 rpmࠉ4Υࠉ28 min) ࡊࠉἸᑏ㔢࡚✨ࢅ㛜ࡄࠉDNA 々⿿࡞ᚪさ࡝ᰶ ⭯⏤ฦࢅྱࢆ࡚࠷ࡾ୯ኳࡡ㯜Ⰵ࠷ᒒࡓࡄࢅ࢙ࢴ࣋ࣤࢺࣜࣆࢲ࣭ࣖࣇ࡞ᅂ཭ࡊࡒࠊ ࡆࡿࢅ㐪ᚨฦ㞫 (13,400 rpmࠉ4Υࠉ12 min) ࡊࠉỷẂࡊࡒ apoptotic ࡝ᒒࢅᣚ ࡔ㎲ࡱ࡝࠷ࡻ࠹࡞ᅂ཭ࡊࡒ୯ኳࡡ㏩᪺࡝ᒒࢅࢻ࢕࣒ࣞࣤࣤࣇ࡚ࣚࣤ℈㐛ࡊࠉ 1/100 ᐖ㔖ࡡ glycerol ࢅຊ࠻ࠉᾦమ✽⣪୯࡞ 20 µl ࡍࡗ⁪ୖࡊ෼⤎ಕᏋࡊࡒࠊ ➠஦⟿ ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ⢥Ꮔᰶ DNA ࡡㄢ⿿ 2.1 ᮞᩩ䛮モⷾ 1) ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ 㞕 (࢜ࢹ࣭㹈࢜࢝ࢠ) 2) MS222 ⁈ᾦ

0.2% (w/v) MS222 (ethyl 3-aminobenzoate methanesulfonate)ࠉ

0.46% (w/v) NaHCO3

3) ࣓࣌ࢩࢻ࢕ࢧ࣭

Potter Elvehjam type Teflon glass homogenizer 4) SuNaSp

0.25 M sucroseࠉ75 mM NaClࠉ0.5 mM spermidineࠉ0.15 mM spermine

5) 3% BSA-SuNaSp

(37)

6) EB 50 mM KClࠉ50 mM HEPES-KOH (pH7.6)ࠉ5 mM MgCl2 ౐⏕├๑࡞2-mercaptoethanol ࢅ῟ຊࡊࡒ (⤂⃨ᗐ 2 mM)ࠊ 7) 30% glycerol-EB 50 mM KClࠉ50 mM HEPES-KOH (pH7.6)ࠉ5 mM MgCl2ࠉ30% glycerol ౐⏕├๑࡞2-mercaptoethanol ࢅ῟ຊࡊࡒ (⤂⃨ᗐ 2 mM)ࠊ 8) ࣉ࢞ࢪࢹ᯹Ⰵᾦ Hoechst33258 ࢅ⤂⃨ᗐ 1 mg/ml ࡞ㄢ⿿ࡊࠉEB ࡚ 500 ಶ࡞ᕵ㔐ࡊ࡙ ౐⏕ࡊࡒࠊ 9) ࣛࢯࣝࢨࢲࣤ

L-α-lysophosphatidylcholine, from Egg Yolk ࢅ⤂⃨ᗐ 2 mg/ml ࡞࡝ ࡾࡻ࠹࡞㉰⣟Ề࡞⁈よࡊ-20Υ࡚ಕᏋࡊࡒࠊ

10) ࢻ࢕࣒ࣞࣤࣤࣇࣚࣤ

30 µm NYLON NET FILTERS NY30 (Millipore) 11) 㐪ᚨᶭ 㐪ᚨᶭTOMY RL-131ࠉ࣭ࣞࢰ࣭TOMY TS-39 2.2 ᪁Ἢ ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ㞕ࢅMS222 ⁈ᾦ࡞ࡻࡽ㯖㓁ࡊࠉ㛜⭙ࡊࡒࡡࡔ⢥ᕛࢅᦤฝ ࡊࡒࠊEB ࢅථࡿࡒࢸ࢔ࢴࢨࣖ࡞⛛ࡊࣅࣤࢬࢴࢹࢅ⏕࠷࡙⾉⟮ࡷ⬙⫣⤄⧂ࢅ⥙㮿 ࡞ཱིࡽ㝎࠷ࡒࠊ᩺ࡒ࡞EB 5 ml ࢅථࡿࡒࢸ࢔ࢴࢨࣖ࡞⢥ᕛࢅ⛛ࡊࠉEB ୯࡚ࣀ ࢦ࣐ࢅ⏕࠷࡙⢥ᕛࢅ࡚ࡀࡾ㝀ࡽ⣵࠾ࡂࡊࡒࠊࡆࡡᠩ⃦ᾦࢅ࣓࣌ࢩࢻ࢕ࢧ࣭࡞⛛

(38)

ࡊࠉ5 ᅂ୕ୖࡌࡾࡆ࡛࡞ࡻࡽᆍୌ࡞ࡊࡒࠊࢻ࢕࣒ࣞࣤࣤࣇ࡚ࣚࣤ℈㐛ᚃࠉ㐪ᚨฦ 㞫 (2,500 rpmࠉ4Υࠉ5 min) ࡊ୕ΰࢅཱིࡽ㝎࠷ࡒࠊࡐࡡ㝷ࠉୖᒒ࡞㉝⾉⌣࠿ỷ Ẃࡊ࡙࠷ࡾሔྙࡢỷẂ୕㒂ࡡⓉ⃦ᒒࡓࡄࢅืࡡ㐪ᚨ⟮࡞⛛ࡊࠉ්ᗐ㐪ᚨฦ㞫ࡊ ࡒ (2,500 rpmࠉ4Υࠉ5 min)ࠊỷẂ∸ࢅᐄῺࡡ SuNaSp 2 ml ࡞ᠩ⃦ࡊࠉࡐࡡᠩ ⃦ᾦ࡞ࣛࢯࣝࢨࢲࣤ_{100 µl ࢅຊ࠻ࠉᐄῺ࡚ 5 ฦ㛣㟴⨠ࡊࡒࠊࢦࣤࣈࣜ 1 µl ࢅࣉ} ࢞ࢪࢹ᯹Ⰵᾦ1 µl ࡛Ίྙࡊࠉ㢟᚜㙶࡚⬲⭯ࢅ☔ヾࡊࡒࠊ⬲⭯ࡈࡿࡒ᯹Ⰵమ࠿ 95% ௧୕࡞࡝ࡾࡱ࡚ࣛࢯࣝࢨࢲࣤฌ⌦ࢅ⧖ࡽ㏁ࡊࡒࠊ⬲⭯ฌ⌦ᚃࡡࢦࣤࣈࣜ࡞ 3% BSA-SuNaSp 2 ml ࢅຊ࠻࡙ 5 ฦ㛣ᨲ⨠ࡊࣛࢯࣝࢨࢲࣤࢅ኶Ὡࡈࡎࡒࠊ㐪ᚨฦ㞫 (2,500 rpmࠉ4Υࠉ5 min) ࡊࡒࡡࡔ୕ΰࢅཱིࡽ㝎ࡀࠉỷẂ∸ࢅࡈࡼ࡞ 3% BSA-SuNaSp 2 ml ࡚ 1 ᅂࠉEB 2 ml ࡚ 2 ᅂὑὯࡊࡒࠊࡆࡡỷẂ∸ࢅ 30% glycerol-EB ࡞ᠩ⃦ࡊ⢥Ꮔᰶ DNA ࡛ࡊࡒࠊ ➠୔⟿ ⤄ᥦ࠻ࢰࣤࣂࢠ㈻ࡡష⿿ ᮇ◂✪࡚⏕࠷ࡒ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻㸝His-Cdt1-Nࠉ His-Cdt1-MࠉHis-Cdt1-C㸞ࠉgemininࠉ࡞ࡗ࠷࡙ࡢ௧ୖࡡ᪁Ἢ࡞ࡻࡽష⿿ࡊࡒࠊ 3.1 His-tag ⼝ྙᆵ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ Cdt1 ᩷∞ࢰࣤࣂࢠ㈻ࡡⓆ⌟࡛⢥ ⿿ 3.1.1 Ⓠ⌟⣌

1) His-Flag-Cdt1-N (pET-15b)ࠉHis-Flag-Cdt1-M (pET-15b)ࠉ

His-Flag-Cdt1-C (pET-15b)

(39)

2) His-geminin (pET-15b) ᙔ◂✪ᐄ὘ᒜᓤ༡ኃ࡞ࡻࡖ࡙ష⿿ࡈࡿࡒHis-tag ⼝ྙᆵ࢓ࣆࣛ࢜ࢵ࣒࢙࢝ࣜ Cdt1᩷∞ࢰࣤࣂࢠ㈻ࢅⓆ⌟ࡌࡾⓆ⌟⏕ࣈࣚࢪ࣐ࢺ࡚࠵ࡾࠊ 3.1.2 ┘Ⓩࢰࣤࣂࢠ㈻ࡡ⢥⿿ Ⓠ⌟⏕ኬ⭘Ⳟࢅࠉ75 µg/ml ampicillinࠉ100 µg/ml chloramphenicol ࢅ῟ຊࡊ ࡒ_{LB ᇰᆀ࡞᳔Ⳟࡊࠉ37Υ࡚⤂እᣲ┖ᇰ㣬ࡊࡒࠊࡆࡿࢅ᩺ࡒ࡝ LB (+ 75 µg/ml} ampicillinࠉ100 µg/ml chloramphenicol) ᇰᆀ (500 ml)࡚10ಶ࡞ᕵ㔐ࡊ37Υ࡚ 1᫤㛣ᣲ┖ᇰ㣬ࡊࡒࡡࡔࠉῺᗐࢅ16Υ࡞ୖࡅࠉ⤂⃨ᗐ0.1 mM ࡡ IPTG ࢅ῟ຊࡊ ࡒࠊ16Υ࡚12᫤㛣ᣲ┖ᇰ㣬ࡊࡒࡡࡔ㐪ᚨࡊ (5,000 rpmࠉ4Υࠉ5 min)ࠉⳞమࢅ ᅂ཭ࡊࡒࠊ ể෫ࡊࡒPBS ࡚ὑὯᚃࠉsonication buffer 10 ml ࢅ῟ຊࡊ࡙㉰㡚ἴ◒◃ࢅ⾔ ࠷ (࣏࢕ࢠࣞࢲࢴࣈࠉᙁᗐ3ࠉ10 sec 㛣㝰࡚20 min)ࠉ㐪ᚨฦ㞫 (17,800 rpmࠉ4Υࠉ 15 min) ࡊ࡙୕ΰࢅᅂ཭ࡊࡒࠊ㉰㡚ἴ◒◃࡛㐪ᚨฦ㞫ࡡ᧧షࢅ⾔ࡖ࡙ᚋࡼࡿࡒ

୕ΰࢅࠉsonication buffer ࡚ᖲ⾦໩ࡊࡒ Ni-NTA 1 ml ࡞ຊ࠻ࠉ4Υ࡚ୌ᫤㛣㌹

಻Ί࿰ࡊࡒࠊࡐࡡᚃࠉࣝࢩࣤࢅ࣑࢜ࣚ࡞ඖሳࡊࠉwash buffer ࡚ὑὯᚃࠉelution

buffer ࢅຊ࠻200 µL/ࣆࣚࢠࢨ࡚ࣘࣤ20ࣆࣚࢠࢨࣘࣤ⁈ฝࡊࡒࠊ྘ࣆࣚࢠࢨࣘࣤ ࡡࢰࣤࣂࢠ㈻ࡡ⃨ᗐࢅῼᏽࡊࠉ㧏⃨ᗐࡡࢰࣤࣂࢠ㈻࠿ᚋࡼࡿࡒࣆࣚࢠࢨࣘࣤࢅ ㏩ᯊࡊࡒࠊ྘buffer ࡡ⤄ᠺࡢ௧ୖࡡ㏳ࡽࠊ ձ His-Flag-Cdt1-NࠉHis-Flag-Cdt1-C sonication buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ1.0% Triton X-100ࠉ

(40)

0.25 mM PMSF wash buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.01% Triton X-100ࠉ 50 mM Imidazole elution buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.01% Triton X-100ࠉ 500 mM Imidazole dialyze buffer 50 mM HEPES-KOH (pH8.0)ࠉ200 mM KClࠉ0.01% Triton X-100ࠉ 5 mM MgCl2 䐗 His-Flag-Cdt1-M sonication buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ1.0% Triton X-100ࠉ 0.25 mM PMSF wash buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.01% Triton X-100ࠉ 50 mM Imidazole elution buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.1% Triton X-100ࠉ 500 mM Imidazole dialyze buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.1% Triton X-100ࠉ 10% sucroseࠉ5 mM MgCl2

(41)

䐘 His-geminin sonication buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ1.0% Triton X-100ࠉ 0.25 mM PMSF wash buffer 50 mM HEPES-KOH (pH8.0)ࠉ300 mM KClࠉ0.1% Triton X-100ࠉ 50 mM Imidazole elution buffer 50 mM HEPES-KOH (pH8.0)ࠉ400 mM KClࠉ0.1% Triton X-100ࠉ 500 mM Imidazole dialyze buffer 50 mM HEPES-KOH (pH8.0)ࠉ400 mM KClࠉ0.1% Triton X-100ࠉ 10% sucroseࠉ5 mM MgCl2 ➠ᄿ⟿㻃 DNA 々⿿Ὡᛮ䛴ῼᏽ 4.1 ᮞᩩ䛮モⷾ 1) Stop C solution 20 mM Tris-HCl pH 7.5ࠉ5 mM EGTAࠉ0.5% SDSࠉ 15 µg/ml ProteinaseK (MERCK) 2) 10% TCA ⁈ᾦ

10% TCA (trichloroacetic acid)ࠉ2% Na4P2O7㹺10H2O

(42)

5% TCAࠉ2% Na4P2O7㹺10H2O 4) e.r

1 M phosphocreatin䚮0.6 mg/ml creatin phospho kinase 5) CHX

10 mg/ml cycloheximide

4.2 ῼᏽ᪁Ἢ

[α-32_{P]dATP ࢅྱࡳ༵᢫ฝᾦ (+ e.rࠉCHX [⃨ᗐ??]) ࡞⢥Ꮔᰶ DNA ࢅ῟ຊࡊࠉ}

23Υ࡚ 90 ฦ࠾ࡼ 180 ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡐࡡᚃࠉ1 ࢦࣤࣈࣜᙔࡒࡽ 160 µl ࡡ Stop C ࢅຊ࠻࡙ DNA 々⿿ཬᚺࢅೳḾࡊࠉ37Υ࡚ 30 min ௧୕࢕ࣤ࢞ࣖ࣊

࣭ࢹࡊ࡙ࢰࣤࣂࢠ㈻ࢅฦよࡈࡎࡒࠊࡆࡿ࡞10% TCA ⁈ᾦࢅ 4 ml ຊ࠻ 4Υ࡚ 30 min ௧୕ཬᚺࡈࡎ࡙ DNA ࢅỷẂࡈࡎࡒࠊཬᚺᾦࢅⰃࡂ᧘ᢶࡊ࡙࠾ࡼ℈⣤࡞ 40 µl ࢪ࣎ࢴࢹࡊࠉࣃ࣭ࢹࣚࣤࣈ࡚ஜ⇩ࡈࡎࡒࠊṟࡽࡡཬᚺᾦࡢ࢝ࣚࢪࣆ࢔ࣜࢰ࣭ ࢅ⏕࠷࡙℈㐛ࡊࠉ10% TCA ⁈ᾦࠉ5% TCA ⁈ᾦࠉ࢙ࢰࢿ࣭࡚ࣜ㡨࡞ὑὯࡊࠉ℈ ⣤࡛ྜྷᵕ࡞ࣃ࣭ࢹࣚࣤࣈ࡚ஜ⇩ࡈࡎࡒࠊஜ⇩ᚃࡆࡿࡼࢅ⣑2 ml ࡡ⣟Ề࡞ථࡿᨲ ᑏὩᛮࢅῼᏽࡊࡒࠊࢲ࢘ࣝࣤࢤࣆක࡞ࡻࡾ32_{P ᨲᑏὩᛮῼᏽࡢᾦమࢨࣤࢲ࣭ࣝࢨ} ࣘࣤ࢜ࢗࣤࢰ࣭ࢅ⏕࠷ࠉ3_{H ࣝࣤࢩ࡚ 1 min ⾔ࡖࡒࠊ℈⣤࠾ࡼࡢධమࡡ 1%ࡡ} [α-32_{P]dATP ࡡᨲᑏὩᛮ࠿ῼᏽ࡚ࡀࠉ࢝ࣚࢪࣆ࢔ࣜࢰ࣭࠾ࡼࡢ DNA ࡞ཱིࡽ㎲ࡱ} ࡿࡒ[α-32_{P]dATP ࡡᨲᑏὩᛮ࠿ῼᏽ࡚ࡀࡾࠊࡆࡿࡼࡻࡽࠉ῟ຊࡊࡒᨲᑏὩᛮࡡ࠹} ࡔࡡర㸚࠿DNA ୯࡞ཱིࡽ㎲ࡱࡿࡒ࠾ࢅ⟤ฝࡊࡒࠊ

(43)

➠஫⟿ ࢠ࣏ࣞࢲࣤ⤎ྙࡡ᳠ฝ

5.1 モⷾ 1) TNIBA

50 mM KClࠉ50 mM HEPES-KOH (pH7.6)ࠉ2 mM MgCl2ࠉ2 mM DTTࠉ

0.5 mM spermidineࠉ0.15mM spermineࠉ2.5mM ATPࠉ 10 µg/ml pepstatin A (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ

10 µg/ml leupeptin (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ0.1% TritonX-100

2) T’NIBA

50 mM KClࠉ50 mM HEPES-KOH (pH7.6)ࠉ2 mM MgCl2ࠉ2 mM DTTࠉ

0.5 mM spermidineࠉ0.15mM spermineࠉ2.5mM ATPࠉ 10 µg/ml pepstatin A (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ 10 µg/ml leupeptin (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ0.01% TritonX-100 5.2 䜳䝱䝢䝅䝷⏤ฦ䛴༟㞫 ࢕࣑ࢿࣇࣞࢴࢹἪ࡞ࡻࡾࢠ࣏ࣞࢲࣤ⤎ྙࢰࣤࣂࢠ㈻ࡡ᳠ฝࢅ⾔࠹ሔྙࠉ⢥Ꮔ ᰶ DNA ࢅ῟ຊࡊࡒ༵᢫ฝᾦ (+ e.rࠉCHX) ࢅ┘Ⓩ࡞ᚺࡋ࡙࢕࣭ࣤ࢞ࣖ࣊ࢹ ࡊ ࡒ ᚃ ࠉTNIBA 1 ml ࢅ ຊ ࠻ ࡙ ㌹ ಻ Ί ࿰ ࡊ ࡒ ࠊ Ί ࿰ ᾦ ࡡ ୖ ࡞ 15%

sucrose-TNIBA ࢅ 100 µl 㔔ᒒࡊ࡙ 9,100×g ࡚ 5 ฦ㛣ࠉ4Υ࡚ swing rotor ࢅ

⏕࠷࡙㐪ᚨฦ㞫ࡊࡒ ࠊ⏲㟻ࡱ࡚୕ΰࢅ㝎࠷ࡒᚃࠉࡈࡼ࡞ TNIBA ࢅ 1 ml ῟

(44)

Ᏸධ࡞㝎ࡀࠉỷẂࡊ࡙࠷ࡾࢠ࣏ࣞࢲࣤ⏤ฦ࡞ sample buffer ࢅຊ࠻࣍ࣜࢷࢴ ࢠࢪࡊࠉ100Υ࡚ 5 min ຊ⇍ࡊ࡙ western blotting ⏕ࢦࣤࣈ࡛ࣜࡊࡒࠊ

༟㞫ࡊࡒࢠ࣏ࣞࢲࣤ⏤ฦࢅࡈࡼ࡞༵᢫ฝᾦ࠽ࡻࡦ⢥⿿ࢰࣤࣂࢠ㈻࡛ࡡ࢕ࣤ ࣭࢞ࣖ࣊ࢨࣘࣤ࡞⏕࠷ࡾሔྙࠉ⢥Ꮔᰶ DNA ࢅ῟ຊࡊࡒ༵᢫ฝᾦࢅ┘Ⓩ࡞ᚺ ࡋ࡙࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉTNIBA 1 ml ࢅຊ࠻࡙㌹಻Ί࿰ࡊࡒࠊΊ࿰ᾦࡡ ୖ࡞15% sucrose-TNIBA ࢅ 100 ml 㔔ᒒࡊ࡙ 5,800×g ࡚ 5 ฦ㛣ࠉ4Υ࡚ swing rotor ࢅ⏕࠷࡙㐪ᚨฦ㞫ࡊࡒ ࠊ㐪ᚨᚃࠉୖᒒࢅࢂࡍ࠾࡞ṟࡊ࡙ࡐࡿ௧አࡡ୕ ΰࢅ㝎ࡀࠉࡐࡡᚃࠉ༵᢫ฝᾦࡱࡒࡢ㐲ᙔ࡝buffer ࡞ᠩ⃦ࡊࡒࠊ ➠ඵ⟿㻃 ඞ␷㝎ཡ 6.1 ᮞᩩ䛮モⷾ

1) rProtein A Sepharose™ Fast Flow (Amersham Biosciences) 2) LFB2/50

40 mM HEPES-KOH (pH 8.0)ࠉ20 mM K-Pi (pH 8.0)ࠉ1 mM EGTAࠉ

10% sucroseࠉ50 mM KClࠉ2 mM DTTࠉ2 mM MgCl2ࠉ

10 µg/ml pepstatin A (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ

10 µg/ml leupeptin (࣋ࣈࢲࢺ◂✪ᡜ)ࠉ2.5 mM ATP

6.2 ᪁Ἢ

rProtein A Sepharose ࢅ⣟Ề࡚ 3 ᅂὑὯࡊࡒᚃࠉ100 mM HEPES-KOH (pH8.0) ࡚ 3 ᅂὑὯࡊࡒࠊSepharose (gel-bed) ࡡ 5 ಶ㔖ࡡඞ␷๑⾉ΰࠉࡵࡊ

(45)

ᢘమࢅ⤎ྙࡈࡎࡒSepharose ࢅ 100 mM HEPES-NaOH (pH8.0)࡚ 3 ᅂὑὯ ࡊࡒᚃLFB2/50 ࡚ 3 ᅂὑὯࡊࡒࠊࢻ࢕࣒ࣞࣤࣤࣇ࡚ࣚࣤ㐪ᚨ℈㐛ࡊ࡙ࣁࢴࣆ ࢒࣭ࢅ࡚ࡀࡾ㝀ࡽ㝎ࡀࠉ㏣ྡྷࡀ࡞㐪ᚨࡌࡾࡆ࡛࡚ Sepharose ࢅᅂ཭ࡊࡒࠊ Sepharose (gel-bed) ࡛➴㔖ࡡ༵᢫ฝᾦࢅຊ࠻ 4Υ࡚ 1 ᫤㛣㌹಻Ί࿰ࡊࡒࠊࢻ ࢕࣒ࣞࣤࣤࣇ࡚ࣚࣤ㐪ᚨ℈㐛 (3,000 rpmࠉ4Υࠉ2 min) ࢅ⾔࠷ࠉ℈ᾦࢅඞ␷ 㝎ཡࡊࡒ༵᢫ฝᾦ࡛ࡊࡒࠊ

➠୏⟿㻃 pull down assay

7.1 ᮞᩩ䛮モⷾ

1) Ni-NTA

2) Gultathione-Sepharose 3) T-LFB2/50

50 mM KClࠉ40 mM HEPES-KOH pH 8.0ࠉ

20 mM potassium phosphateࠉ2 mM MgCl2ࠉ1 mM EGTAࠉ2 mM DTTࠉ10%(w/v) sucroseࠉ1 µg/ml pepstatin Aࠉ1 µg/ml leupeptinࠉ 2.5 mM ATPࠉ0.1% TritonX-100 7.2 ᪁Ἢ 1) His-tag 㐽ᢝᛮ His-tag ࢰࣤࣂࢠ㈻ࢅྱࡳࢰࣤࣂࢠ㈻Ίྙᾦࢅ 23Υ࡚ 20 ฦ㛣࢕࣭ࣤ࢞ࣖ࣊ ࢹࡊࡒᚃࠉT-LFB2/50 ࡚ᖲ⾦໩ࡊࡒ Ni-NTA ࡞ຊ࠻ࠉ4Υ࡚ୌ᫤㛣㌹಻Ί࿰ ࡊࡒࠊࡐࡡᚃࠉ50mM ࡡ imidazole ࢅྱࡳ T-LFB2/50 ࡚ 3 ᅂὑὯᚃࠉsample buffer ࢅຊ࠻⁈ฝࡊࡒࠊ

(46)

2) GST-tag 㐽ᢝᛮ GST-tag ࢰࣤࣂࢠ㈻ࢅྱࡳࢰࣤࣂࢠ㈻Ίྙᾦࢅ 23Υ࡚ 20 ฦ㛣࢕ࣤ࢞ࣖ࣊ ࣭ࢹࡊࡒᚃࠉT-LFB2/50 ࡚ᖲ⾦໩ࡊࡒ Glutathione Sepharose ࡞ຊ࠻ࠉ4Υ ࡚90 ฦ㛣㌹಻Ί࿰ࡊࡒࠊࡐࡡᚃࠉT-LFB2/50 ࡚ 3 ᅂὑὯᚃࠉsample buffer ࢅຊ࠻⁈ฝࡊࡒࠊ ➠ඳ⟿ Licensing assay ⢥ᏄᰶDNA ࢅ῟ຊࡊࡒ༵᢫ฝᾦ (+ e.rࠉCHX) ࡱࡒࡢࣁࢴࣆ࢒࣭ࢅ┘Ⓩ࡞ᚺ ࡋ࡙࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒᚃࠉࡐࡡୌ㒂ࢅ㸧ᮇࡡࢲ࣭ࣖࣇ࡞ฦἸࡊࡒࠊࡐࡡᚃࠉ4 㹳5 ಶ㔖ࡡ[α-32_{P]dATPࠉe.rࠉCHX ࢅྱࡳ༵᢫ฝᾦ࡛Ίྙࡊࡒࠊࡆࡡ࡛ࡀࠉฦ} Ἰࡊࡒ㸧ᮇࡡࢲ࣭ࣖࣇ࡞ࡢࡐࡿࡑࡿ _{geminin ࡡ⤄ᥦ࠻ࢰࣤࣂࢠ㈻ (⤂⃨ᗐ 50} µg/ml)ࢅ῟ຊࡊࡒ༵᢫ฝᾦࡱࡒࡢ geminin ࡡ⤄ᥦ࠻ࢰࣤࣂࢠ㈻ࢅ῟ຊࡊ࡙࠷࡝ ࠷༵᢫ฝᾦࢅຊ࠻ࡒࠊࢦࣤࣈࣜࢅ༵᢫ฝᾦ࡛Ίྙᚃࠉࡈࡼ࡞ 23Υ࡚ 90 ฦ㛣௧ ୕࢕࣭ࣤ࢞ࣖ࣊ࢹࡊࡒࠊࡐࡡᚃࠉDNA ྙᠺ㔖ࡡῼᏽ࡛ྜྷࡋ᪁Ἢ࡚ DNA ࡞ཱིࡽ ㎲ࡱࡿࡒ[α-32_{P]dATP ࡡᨲᑏὩᛮࢅ 100㸚࡛ࡊࠉgeminin ࢅ῟ຊࡊࡒ༵᢫ฝᾦ୯} ࡚ࡡDNA 々⿿Ὡᛮࢅࣚ࢕ࢬࣤࢪ໩Ὡᛮ࡛ࡊ࡙⟤ฝࡊࡒࠊ ➠ஐ⟿ ࢗ࢘ࢪࢰࣤࣇࣞࢴࢹἪ 9.1 ᮞᩩ࡛モⷾ 1) Transfer buffer

DNA複製ライセンス化因子Cdt1の機能ドメインに関する解析