著者
井坂 弘道
学位授与機関
Tohoku University
ฝ ⰾ 㓕 ザ
DNA polymerase δ ࡡࢦ ࣇ ࣗ ࢼ ࢴ ࢹ
Pol31 ࡡ
ᶭ ⬗ ࢺ ࣒ ࣤ ࡞ 㛭 ࡌ ࡾ ゆ ᯊ
᮶ℓኬᏕኬᏕ㝌ⷾᏕ ◂✪⛁ ༡⒬ㄚ⛤๑
⏍ⷾᏕᑍᨯ 㐿ఎᏄⷾᏕฦ㔕 ᆊ ᘧ㐠
┘ ḗ
➠1 ❮ こ ᪠ 4 ➠2 ❮ ᗆ ㄵ 7 ➠1 ⟿ DNA 々⿿࣬ಞᚗࡡណ⩇ 7 ➠ 2 ⟿ DNA polymerase δ 10 ➠ 3 ⟿ Pol δ ࡡࢦࣇࣗࢼࢴࢹ 13 ➠ 4 ⟿ ᮇ◂✪ࡡ┘Ⓩ 19 ➠ 3 ❮ ⤎ ᯕ 20 ➠ 1 ⟿ Pol δ 々ྙమᙟᠺ࡞㛭ࡌࡾࢺ࣒ࣤࡡ᥀⣬ 20 ➠2 ⟿ ⣵⬂⑵る⬗ࢅ࠻ࡾ㡷ᇡࡡ᥀⣬ 24 ➠3 ⟿ 1-113 aa ᩷∞㐛Ⓠ⌟ᰬࡡ⾪⌟ᆵ 29 ➠4 ⟿ 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ⾪⌟ᆵ 31 ➠5 ⟿ PCNA / Mgs1 㐛Ⓠ⌟ࡡᙫ㡢 36 ➠6 ⟿ N ᮆ❻ഁ┞பష⏕㡷ᇡࡡ≁ᏽ 37 ➠ 4 ❮ ⩻ ᐳ 39 ➠ 5 ❮ ᐁ 㥺ᮞ ᩩ ࠽ ࡻ ࡦ᪁ Ἢ 44 ➠ 1 ⟿ ฝⰾ㓕ザ (Saccharomyces cerevisiae) 44 ➠2 ⟿ ᇰᆀ 44 ➠3 ⟿ ࣈࣚࢪ࣐ࢺ 46 ➠ 4 ⟿ DNA ሲᇱ㒼าỬᏽἪ 47 ➠5 ⟿ ⠾᪾ᙟ㈹㌷ᥦἪ 47 ➠ 6 ⟿ Spot assay 47 ➠ 7 ⟿ FACS 48 ➠ 8 ⟿ ࢰࣤࣂࢠ㈹ฝ࠽ࡻࡦ Western blot Ἢ 49 ➠ 9 ⟿ ඞỷ㜾Ἢ 51➠ 10 ⟿ RT-PCR 52 ㅨ ㎙ 54 ཤ ⩻ ᩝ ⊡ 55
ͤ㐿ఎᏄ࣬ࢰࣤࣂࢠ㈹ࡡ⾪エἪ࡞ࡗ࠷࡙
ᮇㄵᩝ࡚ࡢࠉៈౚ࡞ࡊࡒࡖ࡙ࠉฝⰾ㓕ザࡡ㔕⏍ᆵ㐿ఎᏄྞࡢධኬᩝᏊࢰ
ࣛࢴࢠ (e.g. POL31)ࠉን␏ᆵ㐿ఎᏄྞࡢࠉධᑚᩝᏊࢰࣛࢴࢠ (e.g. pol31)ࠉࢰࣤ
ࣂࢠ㈹ྞࡢୌᩝᏊ┘ࡡࡲኬᩝᏊ (e.g. Pol31) ࡚⾪エࡊࡒࠊ
ฦ㓕ザࡡ㐿ఎᏄࡢࠉៈౚ࡞ࡊࡒࡖ࡙ࠉ๑ᑚᩝᏊ⾪エ࡚㔕⏍ᆵ㐿ఎᏄྞࡢ
ྎ⫢࡞
+ࢅ (e. g. cdc27
+)ࠉን␏ᆵ㐿ఎᏄྞࡢྎ⫢࡞-ࢅࡄࡒ (e. g. cdc27
-)ࠊ
㧏➴┷ᰶ⏍∸ࡡ㐿ఎᏄࡢධኬᩝᏊࢰࣛࢴࢠ (e. g. WRNIP1)ࠉ㐿ఎᏄ⏐∸ࡢኬ
ᩝᏊ࡚⾪エࡊࡒ
(e. g. WRNIP1)ࠊ
➠
1 ❮ こ᪠
࠘ ⫴ ᬊ ࠙DNA 々⿿ࣆ࢚࣭ࢠ DNA ഭᐐ➴࡞ࡻࡽೳでࡊࡒ㝷ࠉ᩺⏍㙈⇆⒬ࡡࢼ࣭ࣛࣤࢡ࡞
ࡻࡾഭᐐࡡᅂ㑂ࡷ┞⇆⤄ᥦ࠻ཬᚺ࡞ࡻࡾ々⿿ࡡ්㛜㉫ࡆࡾࠊࡆࡿࡼࡡ㐛⛤࡚రࡼ
࠾ࡡ㝸ᐐ࠵ࡾ࡛Ⰵమࡡ්⥽㉫ࡆࡽࠉⓆࢆࡡⅴᅄ࡛ࡾࠊDNA polymerase δ (Pol
δ) ࡢ DNA 々⿿࡚୯ᚨⓏᙲࢅᢰ࠹㓕⣪࡚ࠉDNA polymerase α/primase 々ྙమࡡྙᠺ
ࡊࡒᩐ༎ሲᇱࡡ▯㙈RNA/DNA ࣈ࣏࣭ࣚࡡఘ㛏ཬᚺࢅ⾔ࡖ࡙࠷ࡾࠊࡆࡡ㓕⣪ࡢ々⿿
ೳでࡊࡒᚃ࡚ࠉ々⿿්㛜ࡈࡿࡾ࡞ࡵ㔔こᶭ⬗ࢅᢰࡖ࡙࠷ࡾࠊฝⰾ㓕ザࡡPol δ ࡢ
3 ࡗࡡࢦࣇࣗࢼࢴࢹࠉPol3ࠉPol31ࠉPol32 ࠾ࡼᵋᠺࡈࡿࠉࡆࡡࣉࢷࣞ 3 㔖మࡡ࠹ࡔ Pol3 ࡢ㓕⣪Ὡᛮࡡᮇమ࡚࠵ࡽࠉPol31 ࡢࡡ 2 ࡗࡡࢦࣇࣗࢼࢴࢹࢅ㏻⤎ࡊࠉPol32 ࡢ DNA ྙ
ᠺ࡞ᚪ㡪PCNA ࡛┞பష⏕ࡌࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾࠊPol3 ࠽ࡻࡦ Pol31 ࢅࢤ࣭ࢺࡌࡾ
㐿ఎᏄࡢḖࡌࡾ࡛⮬ほ࡚࠵ࡾࠉPol31 ࡢ㓕⣪Ὡᛮࡡᮇమ࡚ࡢ࠷ࡡ࡚ㄢ⟿ࢦࣇࣗࢼ ࢴࢹ࡛⩻࠻ࡼࡿࡾࠊPol31 ࡢ Pol δ 々ྙమࡡ々⿿࠽ࡻࡦ々⿿්㛜ࡡᶭ⬗ࡡㄢ⟿ࢅࡊ࡙ ࠷ࡾྊ⬗ᛮ㧏࠷࡞ࡵ࠾࠾ࢂࡼࡍࠉࡆࡿࡱ࡚࡛ࢆ◂✪ࡈࡿ࡙ࡆ࠾ࡖࡒࠊࡐࡆ࡚ᮇ ◂✪࡚ࡢPol31 ࡡ⣵⬂⑵る࡞ᚪ㡪ᶭ⬗ࢅ⌦ゆࡌࡾࡒࡴ࡞ Pol δ ࡡࢦࣇࣗࢼࢴࢹ㛣ࡡ┞ பష⏕࡞ᚪこ Pol31 ࡡࢺ࣒ࣤࡡ≁ᏽࢅ⾔ࡖࡒࠊ ࠘ ⤎ ᯕ ࣬ ⩻ ᐳ ࠙ Pol31 ࡡ࣐ࢿ㓗㒼าࢅ㓕ザ࠾ࡼࣃࢹࡱ࡚セࠉ㧏ᗐ࡞ಕᏋࡈࡿࡒ 10 ࡡ㡷ᇡ (㡷ᇡ I-X) ࡡᏋᅹሒ࿈ࡈࡿ࡙࠷ࡒࠊࡐࡆ࡚ࠉࡆࡿࡼࡡ㡷ᇡࢅ N ᮆ❻࠽ࡻࡦ C ᮆ❻࠾ࡼ㡨ḗ Ḗࡈࡎࡒ⛸ࠍࡡ⣵⬂ᰬࢅష⿿ࡊࠉࡐࡡḖን␏మࡡ⣵⬂⑵る⬗ࡡ᭯↋ࢅㄢࡒࠊ i) Ḗ ᆵ Pol31 ࢅ ⣵ ⬂ ࡞ 㐛 Ⓠ ⌟ ࡌ ࡾ ᇰᆀ࡞࢝ࣚࢠࢹ࣭ࢪࢅ῟ຊࡌࡾࡆ࡛࡞ࡻࡖ࡙㌷ࢅㄇᑙ࡚ࡀࡾGAL1 ࣈ࣓࣭ࣞࢰ࣭ୖ Ὦ࡞⛸Ḗን␏pol31 ࢅථࡊࡒࣈࣚࢪ࣐ࢺࢅ⏕࠷࡙ࠉ⣵⬂⑵る࡞ᚪこ㡷ᇡࢅỬᏽ ࡊࡒࠊࡐࡡ⤎ᯕࠉPol31 ࡡධ㛏 487 ࣐ࢿ㓗ࡡ࠹ࡔࠉ㡷ᇡ I ࡛ II ࡡୌ㒂ࡡࡲࢅⓆ⌟ࡌࡾ N ᮆ❻ഁࡡ 1-113 aa ᩷∞ࢅ㐛Ⓠ⌟ࡌࡾᰬࡢ⏍⫩ྊ⬗࡚࠵ࡖࡒࠊୌ᪁ࠉ㡷ᇡ I ࡡࡲࡡ 1-96
aa ᩷∞ࡷ 108-487 aa ᩷∞ࢅ㐛Ⓠ⌟ࡈࡎࡒ⣵⬂ࡢ⏍⫩࡚ࡀ࠾ࡖࡒࠊPol δ ࡢ DNA ಞ ᚗ࡞ࡵ㛭ࡌࡾࡒࡴࠉ1-113 aa ᩷∞ࢅ㐛Ⓠ⌟ࡌࡾᰬࡡ DNA ഭᐐ࡞ᑊࡌࡾវུᛮࢅ ㄢࡒ࡛ࡆࢀࠉPol31 ධ㛏Ⓠ⌟ᰬ࡛⇆ᵕ࡚࠵ࡖࡒࠊ
ii) Single copy vector ୕ ࠾ ࡼ Ḗ ᆵ Pol31 ࢅ ⣵ ⬂ ࡞ Ⓠ ⌟ ࡌ ࡾ
pol31 㐿ఎᏄ◒ቪᰬࡡ⣵⬂ࡡ⮬ほᛮࢅ Pol31 ࡡ 1-113 aa ᩷∞┞ࡊࡒࡡࡢࠉࡐࡿࢅ
㐛Ⓠ⌟ࡊࡒࡒࡴ࡛⩻࠻ࡼࡿࡒࠊࡐࡆ࡚ ࢣࢿ࣑୕ࡡࣈ࣓࣭ࣞࢰ࣭࣬ࢰ࣭࣐ࢾ࣭ࢰ࣭㒼
าࢅࡵࡗࣈࣚࢪ࣐ࢺ (single copy vector) ࢅ⏕࠷࡙⏍⌦Ⓩ㔖ࡡ Pol31 ࢅⓆ⌟ࡌࡾᐁ㥺
⣌ࢅ⏕࠷࡙⇆ᵕࡡゆᯊࢅ⾔ࡖࡒࠊࡌࡾ࡛ࠉ㐛Ⓠ⌟ࡡሔྙ࡛⇆ᵕ࡞Pol31 1-113 aa ᩷∞
ࢅⓆ⌟ࡌࡾᰬࡢ⏍⫩ྊ⬗࡚࠵ࡽࠉDNA ഭᐐ࡞ᑊࡌࡾវུᛮࡵ㔕⏍ᰬ࡛࡛ࢆ⇆ࡋ
ࡓࡖࡒࠊࡌࢂࡔࠉPol31 ࡡ 1-113 aa ᩷∞ࢅྱࡳࢺ࣒ࣤ I, II ࡡࡲ࡚ Pol31 ࡡᚪ㡪 ᶭ⬗ࢅᢰ࠻ࡾࡆ࡛᪺ࡼ࠾࡛ࡖࡒࠊ
iii) Pol31 1-113 aa ᩷ ∞ Ⓠ⌟ ᰬ ࡡ ⣵ ⬂ ⑵ る ⬗ ࡢ Pol32 ࡞ ౪ Ꮛ ࡊ ࡙ ࠷ ࡾ
1-113 aa ᩷∞Ⓠ⌟ᰬࢅ⏕࠷࡙ࠉࡆࡿࡱ࡚࡞ POL31 ࡛ࡡ㐿ఎᏕⓏ┞பష⏕♟ြࡈࡿ࡙ ࡀࡒ々⿿ᚃಞᚗ࡞㛭ࡌࡾ㐿ఎᏄ࡚࠵ࡾRAD18ࠉMGS1ࠉMMS2 㐿ఎᏄ࠽ࡻࡦ Pol δ ࡡ ࢦࣇࣗࢼࢴࢹࡡୌࡗ࡚ࡵ࠵ࡾ POL32 㐿ఎᏄࡡ◒ቪᰬࢅష⿿ࡊࠉ⣵⬂⑵る⬗ࢅㄢࡒࠊ ࡐࡡ⤎ᯕࠉpol32 㐿ఎᏄ◒ቪ࡞ࡻࡽ 1-113 aa ᩷∞ࡡ㐛Ⓠ⌟ᰬ࠽ࡻࡦ⏍⌦Ⓩ㔖ࡡⓆ⌟ ᰬࡡ࠷ࡍࡿ࡞࠽࠷࡙ࡵ⣵⬂⑵る⬗ࢂࡿࡒࠊ㔕⏍ᆵPol31 ࢅⓆ⌟ࡌࡾᰬ࡚ࡢ pol32 㐿 ఎᏄ◒ቪࢅ⾔ࡖ࡙ࡵ⣵⬂⑵るࡢྊ⬗࡚࠵ࡾࡆ࡛࠾ࡼࠉPol31 ࡡ C ᮆ❻ഁࡢ Pol32 㟸Ꮛᅹ ࡡ⣵⬂ࡡ⏍⫩࡞ᚪ㡪࡚࠵ࡾࡆ࡛♟ြࡈࡿࡒࠊ
iv假 Pol31 N ᮆ ❻ ഁ 1-113 aa ࡢ Pol δ 々 ྙ మ ᙟ ᠺ ࡡ ࡒ ࡴ ࡞ ᚪ こ ࠾ ࡗ ༎ ฦ 㡷 ᇡ ࡚ ࠵
ࡾ
i-iv) ࡡゆᯊ࠾ࡼ Pol31 ࡡ 1-113 aa ࡡ㡷ᇡࡡࡲ࡚ Pol δ ࣉࢷࣞ 3 㔖మᙟᠺࡈࡿࡾྊ⬗
ᛮ⩻࠻ࡼࡿࡒࠊࡐࡆ࡚ࠉHA ࢰࢡࡡ Pol31 1-113 aa ᩷∞ࢅ⣵⬂࡞㐛Ⓠ⌟ࡈࡎࠉ⣵
⬂ฝᾦࢅㄢ⿿ᚃࠉᢘ HA ᢘమࢅ⏕࠷࡙ඞỷ㜾ࢅ⾔ࡖࡒࠊỷ㜾∸୯ࡡ Pol3-FLAG ࡷ
ࡵ࡞᳠ฝࡈࡿࡒࠊୌ᪁ࠉ⣵⬂⏍⫩࡚ࡀ࠷Pol31 1-96 aa ᩷∞࡞ࡢ୦ࢦࣇࣗࢼࢴࢹ࡛ࡵ ⤎ྙ࡚ࡀ࠾ࡖࡒࠊࡌࢂࡔࠉPol31 1-113 aa 㡷ᇡ Pol δ 々ྙమᙟᠺࡡࡒࡴ࡞ᚪこ࠾ࡗ ༎ฦ㡷ᇡ࡚࠵ࡾࡆ࡛ࡢࡋࡴ࡙❟チ࡚ࡀࡒࠊ ௧୕ࡡ⤎ᯕ࠾ࡼࠉධࡂᶭ⬗ᮅ▩࡚࠵ࡖࡒPol31 ࡡ I ࠾ࡼ X ࡡࢺ࣒ࣤࡡ࠹ࡔ N ᮆ❻ ഁ I ࡛ II ⣵⬂ࡡ⏍⫩࡞ᚪ㡪ᶭ⬗ࢅᯕࡒࡌࡆ࡛ࢂ࠾ࡖࡒࠊࡆࡡⓆず Pol31 ࡡ III ࠾ࡼ X ࡡࢺ࣒ࣤ Pol δ 々ྙమࡡᵕࠍᶭ⬗ࢅࡡࡻ࠹࡞ㄢ⟿ࡌࡾ࠾᪺ࡼ࠾࡞ࡌࡾ ◂✪ࡡ❻⥬࡞ࡾࡆ࡛ࢅᙽࡊ࡙࠷ࡾࠊ ᮇ◂✪࠾ࡼࡈࡿࡾPol δ 々ྙమࢦࣇࣗࢼࢴࢹ㛣ࡡ⤎ྙᵕᘟࡡᶅᘟᅒ
➠
2 ❮ ᗆㄵ
➠ 1 ⟿ DNA 々 ⿿ ࣬ ಞ ᚗ ࡡ ណ ⩇ ⣵⬂⑵るࡌࡾࡒࡴ࡞ࡢ㐿ఎᏄࡡᮇమ࡚࠵ࡾ DNA ಶࡌࡾᚪこ࠵ࡽࠉࡆࡡ㐛⛤ࢅ DNA 々⿿࡛࠷࠹ࠊ⣵⬂ࡢࠉG1 →S →G2 →M →G1 ---࡛࠷࠹ࢦࢠࣜࢅᅂࡽ ࡼDNA 々⿿࡛⣵⬂ฦ࡛⧖ࡽ㏁ࡌࠉࡆࡡ⣵⬂࿔ࡡㄢ⟿≤࠹࡛ࠉ⒬ࡷ⩹࡞ࡗ ࡾࠊ 㐿ఎሒࢅᢰ࠹DNA ࡢࠉᏭᏽ࡚࠵ࡾࡆ࡛㔔こ࡚࠵ࡾࠊࡊ࠾ࡊࠉⰅమ DNA ࡢᖏ࡞ኣ ᵕഭࢅུࡄ࡙࠷ࡾࠊDNA ࡞ഭࢅ࠻ࡾ⾪Ⓩこᅄ࡛ࡊ࡙ࡢࠉአⓏࡵࡡ࡛ࡊ࡙ࡢ⣰ አ⥲ࠉහⓏࡵࡡ࡛ࡊ࡙ࡢㅨ㐛⛤࡚⏍ࡍࡾὩᛮ㓗⣪࠵ࡽࠉ⣰አ⥲ࡢࣅ࣐ࣛࢩࣤ 㔖మࢅᙟᠺࡊࠉὩᛮ㓗⣪ࡢ⛸ࠍࡡሲᇱഭࢅㄇⓆࡊࠉࡆࡿࡼࡡഭࡢDNA ࡡ々⿿ࢅ㜴ᐐࡌ ࡾࠊDNA 々⿿㜴ᐐࡈࡿࠉ々⿿ࣆ࢚࣭ࢠೳでࡌࡾ࡛ࠉDNA 㔔㙈ว᩷ 偆double-strand break: DSB假 ⏍ࡋࠉ⣵⬂ほࡷⰅమᏭᏽᛮࢅࡵࡒࡼࡌࠊࡱࡒࠉ≁ょ DNA ྙᠺ㓕⣪ሲᇱ ࡡഭࢅࡽ㉲࠻࡙ྙᠺࢅ⤾ࡄࡾࡆ࡛ (Trans Lesion Synthesis : TLS) ࡵ㉫ࡆࡾ ࠉࡐࡡ㝷ࠉ ഭࡡ⛸㢦ࡷ⏕ࡈࡿࡾDNA ྙᠺ㓕⣪࡞ࡻࡖ࡙ㄏࡖࡒሲᇱཱིࡽ㎰ࡱࡿ࡙✲↓ን␏ࢅ⏍ࡋࠉ ⒬ࡷ⩹ࡡⅴᅄ࡛ࡾࡆ࡛ࡵ࠵ࡾࠊୌ᪁ࠉࡆࡡࡻ࠹රྙࢅ㜭ࡃࡒࡴ࡞ࠉ⏍∸ࡢ㐿ఎሒࢅと☔࡞⥌ᣚࡌࡾࡒࡴᵕࠍᶭ ⬗ࢅങ࠻࡙࠷ࡾࠊౚ࠻ࡣ DNA ࡡ㔔㙈ว᩷࡞ࡗ࠷࡙ࡢ┞⇆⤄ࡲᥦ࠻ಞᚗ 偆Homologous Recombination: HR假 ࡷ㟸┞⇆ᮆ❻⤎ྙಞᚗ 偆Non-Homologous End-Joining: NHEJ假 ࡛࠷ࡖ ࡒᶭᵋ഼ࡀࠉ㔔㙈ว᩷࡞ࡻࡾⰅమᏭᏽࢅ㜭࠷࡚࠷ࡾ (Fig.0)ࠊ
ୌౚ࡛ࡊ࡙ࠉ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔ࡡ␏ᖏ࡛㐿ఎࡷ⒬ࡡⓆ⌟࡛ࡡ㛭㏻ᛮࢅ♟ࡊࡒሒ࿈ࢅ ᣪࡅࡾ (Lee et al., 2007)ࠊPelizaeus-Merzbacher Disease (PMD) ࡢ࣐࢙ࣛࣤᙟᠺධ࣬⢥♼㐘 ິⓆ㐡㐔࣬ᛮᄿ⫝㯖⑯ࢅᮮࡌX ㏻㙈㐿ఎᛮ⑄ᝀ࡚࠵ࡽࠉࡐࡡ 60-70% ptoteolipid protein 1 (PLP1) ࡡ㐿ఎᏄ㔔々࡞⏜ᮮࡌࡾ Plp1 ࡡ㐛Ⓠ⌟࡞ࡻࡖ࡙Ⓠࡌࡾࡆ࡛▩ࡼࡿ࡙࠷ ࡾࠊࡆࡡᝀ⩽ࡡ㐿ఎᏄ㒼าࢅゆᯊࡊࡒ⤎ᯕࠉୌ㒂ࡡᝀ⩽࠾ࡼmicrohomology ࡛ࡣࡿࡾ㐿ఎ Ꮔ୕ࡡᩐሲᇱ⛤ᗐࡡ┞⇆㒼าࢅ⑧⏲࡞ PLP1 㐿ఎᏄ࿔㎮ࡡ㒼า᩷⤾Ⓩ࡞㔔々ࡊ࡙୩ࡩ≁
ᚡⓏ㒼าⓆずࡈࡿࡒࠊⰅమ୕ࡡ PLP1 ࿔㎮࡞ࡢ々⿿ࣆ࢚࣭ࢠೳでࡊࡷࡌ࠷々㞟 DNA ᵋ㏸ࢅ࡛ࡾࡆ࡛ࢅ♟ြࡌࡾ㒼าᏋᅹࡊ࡙࠷ࡒࡆ࡛࠾ࡼࠉⴥ⩽ࡼࡢࡆࡡࡻ࠹Ⰵమ ් ⥽ ᠺ ㉫ ࡆ ࡾ ฦ Ꮔ ࣒ ࢜ ࢼ ࢫ ࣑ ࡡ ࣓ ࢸ ࣜ ࡛ ࡊ ࡙ FoSTeS 偆Fork Stalling and Template Switching假 ࢅᥞฝࡊ࡙࠷ࡾ (Fig. 1)ࠊࡆࡡ࣓ࢸ࡚ࣜࡢ々⿿ࣆ࢚࣭ࢠࡡ㐅⾔㗢ᆵ㙈୕ࡡ ഭ➴࡞ࡻࡖ࡙ೳでࡊࡒࠉmicrohomology ࢅࡊ࡙々⿿ࣆ࢚࣭ࢠ㗢ᆵ㙈୕ࢅ๑ᚃ࡞ࢱࢻ ࣐ࢴࢠ࡞ࢪ࢞ࢴࣈࡊࠉ⤎ᯕⓏ࡞PLP1 㐿ఎᏄ㔔々ࡊ࡙々⿿ࡈࡿࡾࠊࡆࡡࡻ࠹࣒࢜ࢼࢫ࣑ ࢅࡊ࡙Ⰵమࡡ්⥽ᠺ㉫ࡆࡽᚋࡾ࡛࠷࠹ࡆ࡛ࡢ㔔々㐿ఎᏄ࡞ࡻࡾ㐿ఎ࡛࠷࠹㝀ࡼࡿ ࡒ⑄ᝀࡓࡄ࡚ࡢࡂࠉ㐿ఎᏄࡡን␏࡞㉫ᅄࡌࡾ⒬ࢅྱࡳ⛸ࠍࡡ⑄ᝀࡡⅴᅄ々⿿ࣆ࢚ ࣭ࢠࡡ㐅⾔࣬ೳで࡞ᐠ࡞㛭౿ࡊ࡙࠷ࡾࡆ࡛ࢅ♟ြࡊ࡙࠷ࡾࠊ
Fig. 0 Biological phenomenon related to DNA replication and repair. The protection of genomic integrity is a major challenge for living cells that are continuously exposed to endogenous and environmental DNA-damaging insults. To cope with the consequences of DNA lesions which interfere with DNA replication, cells are equipped with an efficient defense mechanism termed the DNA damage response.
Fig.1 A model of FoSTeS (a) FoSTeS Model (Lee et al., 2007), (i) One replication fork (dark blue and red, solid lines) with a lagging strand (red, dotted line) would invade a second fork (purple and green, solid lines), followed by (ii) DNA synthesis (green, dotted line). After the fork disengages (iii), the original fork (dark blue and red, solid lines) with its lagging strand (red and green, dotted lines) could invade a third fork (gray and black, solid lines). Dotted lines represent newly synthesized DNA. Serial replication fork disengaging and lagging strand invasion could occur several times before (iv) resumption of replication on the original template (b) Fork Repair and Chromosomal Rearrangements by Template Switching (Branzei et al., 2007) Replication forks encountering DNA lesions (red star) on the template strand can engage the nascent chains into template switching to bypass the DNA lesion. Forks encountering low-copy repeats or repetitive elements are prone to stalling and, occasionally, switch templates in the presence of a nearby template at another fork, thus generating chromosomal rearrangements. This process might require regions of microhomology (red bars).
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2⟿ DNA polymerase δ
DNA polymerase δ (Pol δ)ࡢ 1976 ᖳ࡞ࢦ࢟ࡡ㦭㧂⣵⬂ࡻࡽ┷ᰶ⏍∸ࡡ 4 ␊┘ࡡ DNA ྙ ᠺ㓕⣪࡛ࡊ࡙Ⓠずࡈࡿࡒ (Byrnes et al., 1976)ࠊࡆࡡ㓕⣪ࡢࡐࡿࡱ࡚┷ᰶ⣵⬂ࡡこ DNA ྙᠺ㓕⣪࡛⩻࠻ࡼࡿ࡙࠷ࡒDNA polymerase α (Pol α) ࡞ࡢ↋࠷ 3’→5’࢙࢞ࢮࢽࢠ࣭ࣝࢭὩ ᛮࢅࡵࡗ㓕⣪࠵ࡾࡆ࡛☔ヾࡈࡿࡒࠊPol δ ࡢࡊࡣࡼࡂࡡ㛣ࠉPol α ࠾ࡼὬ⏍ࡊࡒ々ྙమ࡚࠵ ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡒࠊࡐࡡᚃࠉPol δ ༟≺࡚ࡢᩐሲᇱࡡ▯࠷ DNA ྙᠺࡊ࠾⾔࠻࠷ࡡ࡞ᑊࡊࠉ S ࡡᰶ࡞ᏋᅹࡌࡾฦᏄ㔖 37 kDa ࡡࢰࣤࣂࢠ㈹ࢅಀ㐅ᅄᏄ࡛ࡊ࡙῟ຊࡌࡾࡆ࡛࡞ࡻࡽࠉ DNA ྙᠺࡡᣚ⤾ᛮ୕᪴ࡌࡾࡆ࡛᪺ࡼ࠾࡞ࡈࡿࡒ (Tan et al., 1986)ࠊ1987 ᖳ࡞ࡆࡡಀ㐅 ᅄᏄProliferating Cell Nuclear Antigen (PCNA) ࡚࠵ࡽࠉム㥺⟮හ࡚ࡡ SV40 ࣜࢪࡡ DNA 々⿿ཬᚺ࡞PCNA ᚪ㡪࡚࠵ࡾࡆ࡛♟ࡈࡿࡒࠊࡆࡡࡻ࠹⤊⦃࡚ Pol δ ᩺ࡒ DNA 々 ⿿㓕⣪࡛ࡊ්࡙Ⓠずࡈࡿࡾࡆ࡛࡛ࡖࡒ (Prelich et al., 1987)ࠊ
Fig. 2 Eukaryotic replication fork. (Garg et al., 2005) The minimal set of proteins for fork propagation are indicated.
DNA ࡡ々⿿㐛⛤࡚ࡢ Pol α/primase RNA ࣈ࣏࣭ࣚࢅྱࡴ࡙ᩐ༎ࢽࢠ࢛ࣝࢲࢺࡡ▯㙈 DNA ࢅྙᠺࡌࡾ (Waga et al., 1998; Wang et al., 1991)ࠊPol α DNA ྙᠺࢅೳでࡌࡾ࡛ࠉࣈ ࣏࣭ࣚࡡ3’ᮆ❻࡞ Replication factor C (RFC) ౪ᏋⓏ࡞ PCNA ⤎ྙࡌࡾࠊPol δ ࡢ PCNA ౪ᏋⓏ࡞DNA ࡞࣭ࣞࢸࣤࢡࡈࡿࠉPol α ࡡྙᠺࡊࡒ▯㙈 DNA ࡡఘ㛏ཬᚺࢅ⾔࠹ࡆ࡛࡞ࡻ ࡽlagging 㙈ྙᠺࡈࡿࡾࠊ▯㙈 DNA ఘ㛏ࡊࠉ㝼ࡌࡾ RNA ࣈ࣏࣭ࣚ࡞㐡ࡌࡾ࡛ RNA ࣈ࣏࣭ࣚࡢRNase H ࠽ࡻࡦ Fen-1 ࡞ࡻࡽཱིࡽ㝎࠾ࡿࡾࠊࡈࡼ࡞ DNA ligase 1 㝼ࡌࡾᒱ ᓧࣆࣚࢡ࣒ࣤࢹࢅ㏻⤎ࡊࠉDNA lagging 㙈ࡡྙᠺ㐅⾔ࡊ࡙࠷ࡂ (Fig. 3)ࠊ
⌟ᅹࡱ࡚࡞ࠉDNA 々⿿㓕⣪࡞㛭ࡌࡾ㓕ザࡡ㐿ఎᏕⓏゆᯊࠉ࠽ࡻࡦࡐࡿࡑࡿࡡ DNA polymease ࡡ⏍ᏕⓏ≁ᛮ࡞㛭ࡌࡾゆᯊ࠾ࡼࠉ⣵⬂හ࡚Ⰵమࢣࢿ࣑ࡡ々⿿࡞├㛭ࡌࡾ ࡡࡢPol αࠉPol δࠉPol ε ࡡ 3 ⛸㢦࡚࠵ࡾࡆ࡛♟ࡈࡿ࡙ࡀࡒࠊPol δ ࠽ࡻࡦ Pol ε ࡡ DNA 㙈 ఘ㛏ཬᚺࡡᙲฦᢰ࡞ࡗ࠷࡙ࡢㅎㄕᏋᅹࡌࡾࠉౚ࠻ࡣࠉࢨࡡ⬒⭚࠾ࡼ⢥⿿ࡊࡒPol αࠉPol δࠉPol ε ࡡὩᛮࢅム㥺⟮හ࡚セ㍉ࡊࡒᐁ㥺࡚ࡢࠉPol ε PCNA 㟸Ꮛᅹ࡚ࡵ 1000 ሲᇱ௧୕ࡡఘ㛏ཬᚺࢅ⾔࠹ࡆ࡛࡚ࡀࡾࡡ࡞ᑊࡊࠉPol δ ࡢࠉPCNA Ꮛᅹ࡞ࡢ Pol ε ࡛⇆ᵕ ࡞1000 ሲᇱࡡ㛏ࡈࡡ DNA ྙᠺࡌࡾࡵࡡࡡࠉPCNA Ꮛᅹࡊ࠷࡛ࠉPol α ྙᠺࡌࡾ DNA ࡻࡽࡵ▯࠷DNA ࡊ࠾ྙᠺ࡚ࡀ࠷࡛࠷࠹⤎ᯕᚋࡼࡿ࡙࠷ࡾ (Weiser et al., 1991)ࠊࡆࡡࡻ ࠹࡞ࠉPol δ ࡡఘ㛏ཬᚺࡢᏰධ࡞ PCNA ࡞౪Ꮛࡊ࡙࠽ࡽࠉ༟≺࡚ࡡ DNA ྙᠺཬᚺࡡᣚ ⤾ᛮࡢPol δ ࡻࡽ Pol ε ඁࡿ࡙࠷ࡾࡒࡴࠉPol δ ࡞ lagging 㙈ࡡྙᠺࢅ⾔࠷ࠉࡻࡽᣚ⤾ᛮ ࡡ㧏࠷Pol ε leading 㙈ࡡྙᠺࢅ⾔࠹࡛࠷࠹ㄕ᭯ງちࡈࡿ࡙ࡀࡒࠊࡱࡒ᭩㎾ࠉฝⰾ㓕ザࢅ ⏕࠷ࡒゆᯊ࠾ࡼleading 㙈ࡡྙᠺ࡞ Pol ε 㛭ࡌࡾࡆ࡛♟ြࡈࡿ (Pursell et al., 2007)ࠉPol δࠉPol ε ࠍlagging 㙈ࠉleading 㙈ࡡྙᠺࢅ⾔࠹࣓ࢸࣜᥞฝࡈࡿ࡙࠷ࡾࠊࡊ࠾ࡊࡼࠉ SV40 ࡡ DNA ム㥺⟮හ々⿿⣌࡚ࡢ Pol αࠉPol δ ࡡ 2 ⛸㢦ࡡ DNA ྙᠺ㓕⣪࡚ DNA 々⿿Ᏸ ࡌࡾࡆ࡛ࡷࠉPol ε ࡡ࣒࣭࣎ࣛࣚࢭࢺ࣒ࣤฝⰾ㓕ザࡡ⣵⬂⑵る࡞ᚪ㡪࡚ࡢ࠷ࡆ࡛ࡵ ♟ࡈࡿ࡙࠽ࡽ (Kesti et al., 1999)ࠉPol ε ࡡ㓕⣪ὩᛮࢅḖࡊࡒ᮪௲ୖ࡚ࡢ leading 㙈ࡡྙᠺࡵ Pol δ ᢰᙔࡌࡾࡡ࡚ࡢ࠷࠾࡛⩻࠻ࡼࡿ࡙࠷ࡾࠊࡊࡒࡖ࡙ࠉDNA 々⿿࡞㛭ࡌࡾ DNA ྙᠺ㓕⣪ࡡ㐽ᢝᛮ࡞ࡢ࠵ࡾ⛤ᗐࡡᙆງᛮ࠵ࡽࠉୌࠉPol α ࣈ࣏࣭ࣚࢅྙᠺࡌࡾ࡛㒌 ྙࡡⰃ࠷DNA ྙᠺ㓕⣪㐽ᢝࡈࡿࠉධమࡡ々⿿ࢅ⾔ࡖ࡙࠷ࡂࡻ࠹࡚࠵ࡾࠊ
DNA ྙᠺཬᚺࡢ࡞ DNA 々⿿࡞こヴࡈࡿࡾࠉኣᵕ DNA ಞᚗཬᚺࠉ⤄ࡲᥦ࠻ཬᚺ ࡞࠽࠷࡙ࡵࠉࡐࡡ㏭୯ࡡ㐛⛤࡚⏍ࡋࡾDNA ࢟ࣔࢴࣈࢅᇔࡴࡾ㝷࡞ DNA ྙᠺᚪ㡪࡛ࡾࠊ ࡆࡡDNA ࢟ࣔࢴࣈࢅᇔࡴࡾ DNA ྙᠺࡵ 3’ᮆ❻࠾ࡼຝ⋙ࡻࡂ㛜╂ࡈࡿࡾᚪこ࠵ࡽࠉ3’ᮆ ❻ࡡㆉืὩᛮࢅ᭯ࡌࡾRFC 々ྙమ࡛ PCNA ࡛࡞౪Ꮛࡌࡾ Pol δ ࡢࠉPol ε ࡛භ࡞こಞᚗ DNA ྙᠺ㓕⣪࡛ࡊ഼࡙ࡂࠊࡱࡒࠉฝⰾ㓕ザࡡ Pol δ ࢅࢤ࣭ࢺࡌࡾ㐿ఎᏄࡡን␏ᰬࡡゆᯊࠉ⢥ ⿿ࢰࣤࣂࢠ㈹࡞ࡻࡾಞᚗཬᚺࡡ්ᵋ⠇ࡡ◂✪࡞ࡻࡽࠉࡆࡿࡼࡡ㓕⣪ࢽࢠ࢛ࣝࢲࢺ㝎ཡಞ
ᚗࠉሲᇱ㝎ཡಞᚗࠉ࣐ࢪ࣏ࢴࢲಞᚗࠉDNA ᮇ㙈ว᩷ಞᚗ࡞࠽࠷࡙㔔こᙲࢅᢰ࠹ࡆ࡛ ♟ࡈࡿ࡙࠷ࡾ (Wang et al., 2004; Burgers et al., 1998)ࠊDNA 々⿿⇆ᵕࠉࡆࡿࡼࡡಞᚗཬᚺ ࡡኣࡂ࡞ࡗ࠷࡙ࡵPCNA 㛭ࡌࡾࡆ࡛ࡢ᪺☔࡚࠵ࡾࠉᮅࡓ Pol αࠉδࠉε ࡡ࠷ฦࡄ࡞㛭 ࡊ࡙ࡢ᪺Ⅴኣ࠷ࠊPol δ ࡞㛭ࡊ࡙ࡢࠉDNA 々⿿࡞ഭࡽ㉲࠻ᆵࡡ DNA ྙᠺ㓕⣪ ࡛භ⇆ࡊ࡙DNA ഭ㒂నࡡ DNA ྙᠺ࡞㛭ࡌࡾ⤎ᯕࡵᚋࡼࡿ࡙࠷ࡾ (Gerik et al., 1998)ࠊ
Fig. 3 The lagging strand synthesis (Garg et al., 2005) Initially, Pol α/primase complex synthesizes RNA/DNA primer. The processive RFC/PCNA/pol δ complex then extends the nascent DNA strands to form the continuously synthesized lagging strand complex.
➠
3 ⟿ Pol δ ࡡࢦࣇࣗࢼࢴࢹ
ິ∸⣵⬂࠾ࡼ⢥⿿ࡈࡿࡒPol δ ࡢᙔิࠉࣉࢷࣞ㔖మ࡚ࠉฦᏄ㔖 125 kDa ࡡよ፳ࢦࣇࣗࢼ ࢴࢹ࡛⣑50 kDa ࡡᶭ⬗ᮅ▩ࡡࢦࣇࣗࢼࢴࢹ࠾ࡼᵋᠺࡈࡿࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡒࠉ㓕ザࡡ⢥ ⿿ᵾဗࡡゆᯊ࠾ࡼฝⰾ㓕ザ࡞࠽࠷࡙Pol δ ࡢ 3 ⛸ࡡࢦࣇࣗࢼࢴࢹ࠾ࡼᵋᠺࡈࡿ࡙࠷ࡾࡆ࡛ ♟ࡈࡿࡒ (Burgers et al., 1998)ࠊୌ᪁ࠉฦ㓕ザ࠾ࡼ⢥⿿ࡈࡿࡒ Pol δ ࡛⇆ᵕ࡞ࠉࢨࡡ⬒⭚ࠉ ࣃࢹ࠾ࡼ⢥⿿ࡈࡿࡒ Pol δ ࡢ 4 ⛸ࡡࢦࣇࣗࢼࢴࢹ࠾ࡼᵋᠺࡈࡿ࡙࠷ࡾࡆ࡛♟ࡈࡿࡒ (Zuo et al., 1997; Liu et al., 2000; MacNeill et al., 1996) (Table 1)ࠊࡆࡿࡼࡡࢦࣇࣗࢼࢴࢹࢅⓆ⌟ ࡌࡾࡆ࡛࡚ᶭ⬗Ⓩ࡞༎ฦࣃࢹPol δ 々ྙమࢅ්ᵋ⠇࡚ࡀࡾࡆ࡛࠾ࡼࠉࣉࢷࣞ 4 㔖మᆵဳ ິ∸Pol δ ࡡᮇᮮࡡጶ࡚࠵ࡾ࡛⌟Ⅴ࡚ࡢ⩻࠻ࡼࡿ࡙࠷ࡾ (Podust et al., 2002) (Fig. 5) ࠊ ฝ ⰾ 㓕 ザ ࡡ ࢦ ࣇ ࣗ ࢼ ࢴ ࢹ
i) Large subunit (Pol3)
ฝⰾ㓕ザ࡞࠽࠷࡙よ፳ࢦࣇࣗࢼࢴࢹࡢ POL3 ࡞ࢤ࣭ࢺࡈࡿࠉ㐿ఎᏄ⏐∸ࡡ Pol3 ࡢ⣵⬂⑵ る࡞ᚪ㡪࡚࠵ࡾࠊPOL3 ࡡ࣓࣌ࣞࢡ࡞࠽࠷࡙ࡢ⛸ࢅ㉰࠻࡙㧏࠷ಕᏋᛮ࠵ࡽࠉࣃࢹ࡞࠽ࡄࡾ よ፳ࢦࣇࣗࢼࢴࢹp125 ࡛ࡵ 50偃⛤ᗐ࣐ࢿ㓗㒼าୌ⮬ࡌࡾࠊPol3 ࡢ୯ኳ㒂࡞࣒࣭࣎ࣛࣚ ࢭὩᛮࡡ࠾3’→5’࢙࢞ࢮࢽࢠ࣭ࣝࢭὩᛮࡵ᭯ࡊࠉᰧとᶭ⬗࡞ࡻࡾ fidelity ࡡ㧏࠷ DNA 々 ⿿ࢅ⾔࠹ࠊࡱࡒࠉኬ⭘ⳞࡡDNA 々⿿㓕⣪ࠉPol II ࡞㢦జࡊࡒ࣐ࢿ㓗㒼าࢅࡵࡗࡆ࡛࠾ࡼ B family ࡡ DNA 々⿿㓕⣪ࡡୌࡗ࡞ฦ㢦ࡈࡿࠉᵋ㏸ゆᯊࡢࡈࡿ࡙࠷࠷ࠉ࣐ࢿ㓗㒼าࡡ 㢦జᛮ࠾ࡼ࠽ࡐࡼࡂDNA ྙᠺ㓕⣪ࡡᵋ㏸࡞࠽࠷࡙ཿⓏ Palm-Finger-Thumb ᵋ㏸ࢅ࡛ࡖ ࡙࠷ࡾࡓࢀ࠹࡛᥆ῼࡈࡿ࡙࠷ࡾ (Fig. 4)ࠊฝⰾ㓕ザ࡞࠽࠷࡙ࡢ 3’→5’࢙࢞ࢮࢽࢠ࣭ࣝࢭὩ ᛮࢅࢤ࣭ࢺࡌࡾ㡷ᇡࡡን␏࡞ࡻࡖ࡙⮤↓Ⓠ⏍Ⓩ✲↓ን␏⋙ኬࡀࡂ୕᪴ࡌࡾࡆ࡛▩ࡼ ࡿ࡙࠷ࡾ (Simon et al., 1991)ࠊࡱࡒࠉ࣏ࢪ Pol3 ࡡ࢙࢞ࢮࢽࢠ࣭ࣝࢭὩᛮ࡞ᑊᚺࡌࡾ࣐ ࢿ㓗࡞ን␏ࢅᑙථࡊࡒゆᯊ࡚ࡢ⭐⑾ᙟᠺ⋙ࡡ㢟ⴥ୕࡛᪴ࠉᑋࡡ▯⦨ሒ࿈ࡈࡿ࡙࠷ࡾ (Goldsby et al., 2001; Goldsby et al., 2002)ࠊࡱࡒࠉ࣒࣭࣎ࣛࣚࢭ㡷ᇡࡡን␏࡞ࡻࡾ࣏ࢪࡡ⭐ ⑾ᙟᠺ⋙ࡡ୕᪴ࠉᑋࡡ▯⦨ࠉⰅమᏭᏽᛮࡡⓆ⌟ሒ࿈ࡈࡿ࡙࠽ࡽࠉࡆࡿࡼࡡሒ࿈࠾ ࡼPol δ ࡡᶭ⬗ࡡንㄢ࡛Ⓠ࢝ࣤࡡ㛭౿♟ြࡈࡿ࡙࠷ࡾ (Venkatesan et al., 2007)ࠊ
ࢦࣇࣗࢼࢴࢹ㛣ࡡ┞பష⏕࡞ࡗ࠷࡙ࡢ Pol31 ࡛⤎ྙࡊ࡙࠽ࡽࠉࡆࡡ┞பష⏕࡞ࡢ⣵⬂හࠉ ム㥺⟮හ࡛ࡵ࡞Pol3 ࡡ C ᮆ❻㒂࡞Ꮛᅹࡌࡾ C4 Zn finger 㡷ᇡᚪこ࡚࠵ࡾࡆ࡛ሒ࿈ࡈࡿ ࡙࠷ࡾ (Sanchez Garcia et al., 2004)ࠊࡊ࠾ࡊࠉZn finger 㡷ᇡࡡ⏍ᏕⓏᙲ࡞ࡗ࠷࡙ࡢ᪺ ࡼ࠾࡚ࡢࡂࠉࡆࡡ⤎ྙࡡ⣵⬂⑵る࡞ᑊࡌࡾណ⩇ࡢࡻࡂฦ࠾ࡖ࡙࠷࠷ࠊୌ᪁ࠉฦ㓕ザ ࡡ࣓࣌ࣞࢡ࡚ࡢࡆࡡ㡷ᇡࡡḖ࡞ࡻࡖ࡙⣵⬂⑵るೳでࡌࡾࡆ࡛ずฝࡈࡿ࡙࠷ࡾࠊࡱࡒࠉ Pol3 ࡡࣃࢹ࣓࣌ࣞࢡ࡚ࡢム㥺⟮හ࡚㛏ⱖ⟿ PCNA ࡛┞பష⏕ࡌࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾࠊ
Fig. 4 Structure of B-family DNA polymerase. Pol δ belongs to the B class of DNA polymerases (Burgers et al., 2001). Structural information about this class of enzymes derives from distantly related cousins, i.e., from bacteriophage RB69 (Hopfner et al., 1999). ii) Second subunit (Pol31)
ฝⰾ㓕ザ࡞࠽࠷࡙➠2 ࡡࢦࣇࣗࢼࢴࢹࡢ POL31/HYS2/SDP5 ࡞ࢤ࣭ࢺࡈࡿ࡙࠽ࡽࠉ㐿ఎ Ꮔ⏐∸ࡡ Pol31 ࡵ Pol3 ࡛⇆ᵕ࡞⣵⬂⑵る࡞ᚪ㡪࡚࠵ࡾ (ฦ㓕ザࡡ Pol31 ࣓࣌ࣞࢡ࡚࠵ࡾ cdc1+ࡵ⣵⬂⑵る࡞ᚪ㡪࡚࠵ࡾ)ࠊPOL31 㐿ఎᏄࡡ DNA 々⿿ࡡ㛭ࡢᙔิࠉࡐࡡⅤን␏ sdp5-1 POL3 ࡡῺᗐវུᛮን␏ᰬ pol3-14ࠉpol3-11 ࡡῺᗐវུᛮࢅᢒโࡌࡾࡆ࡛࡞ࡻࡖ࡙ ずฝࡈࡿࡒ (Giot et al., 1995)ࠊࡱࡒࠉPol31 ࡡ 170 ␊┘ࡡ࣐ࢿ㓗ࡡ࣐ࢪࢬࣤࢪን␏ᰬ hys2-1 Ribonucleotide Reductase (RNR) 㜴ᐐ࡚࠵ࡾ Hydroxyurea (HU) ࡡវུᛮࢪࢠ࣭ࣛࢼࣤࢡ ࠾ࡼずฝࡈࡿ (Sugimoto et al., 1995)ࠉ᭩⤂Ⓩ࡞ࡢࡆࡡ㐿ఎᏄ⏐∸ Pol δ ࡡࢦࣇࣗࢼࢴࢹࢅ ࢤ࣭ࢺࡊ࡙࠷ࡾࡆ࡛᪺ࡼ࠾࡞ࡖࡒ (Hashimoto et al., 1998)ࠊୌ⯙Ⓩ࡞ Pol31 ࡢ Pol3 ࡛ Pol δ 々ྙమࡡࢤࢅᙟᠺࡌࡾ࡛⩻࠻ࡼࡿ࡙࠽ࡽࠉᑛࡂ࡛ࡵム㥺⟮හ࡚ࡢฝⰾ㓕ザ Pol3-Pol31 ࡡࣉࢷࣞ㔖మ࡚ࡵࠉ㐛㔖ࡡ PCNA ῟ຊࡵࡊࡂࡢࡆࡡࣉࢷࣞ㔖మ㐛㔖Ꮛᅹࡊࡒ
࡞ࡢ༎ฦఘ㛏ཬᚺびᐳࡈࡿࡒ࡛࠷࠹ሒ࿈ࡈࡿ࡙࠷ࡾ (Burgers et al., 1998)ࠊࡊ࠾ࡊ ࡼࠉPol31 ࡛ PCNA ࡡ├ࡡ⤎ྙࡢ⌟ᅹࡡ࡛ࡆࢀ☔ヾࡈࡿ࡙࠷࠷ࠊୌ᪁ࠉࣃࢹࡡ Pol31 ࣓࣌ࣞࢡࡡゆᯊ࡚ࡢࠉPol δ ࡡఘ㛏ཬᚺಀ㐅࡞ࡢ Pol32 ࣓࣌ࣞࢡ㟸Ꮛᅹୖ࡚ࡢ Pol31 ࣓࣌ࣞࢡ ᚪこ࡚࠵ࡾࡆ࡛♟ࡈࡿ࡙࠷ࡾ (Zhou et al., 1997)ࠊࡱࡒࠉPol31 ࡡ 60-68 aa ࡞┞ᙔࡌࡾ࣓ ࢲ࣭ࣆ࡚ࡡPCNA ࡡ⤎ྙሒ࿈ࡈࡿ࡙࠽ࡽ (Lu et al.,2002)ࠉฝⰾ㓕ザ࡞࠽࠷࡙ POL32 㐿ఎ Ꮔᚪ㡪㐿ఎᏄ࡚ࡢ࠷ࡆ࡛ࢅ⩻៎ࡌࡾ࡛ࠉPol32 Ꮛᅹࡊ࠷࡞ࡢࠉPol3-Pol31 ࣉࢷࣞ 2 㔖మ࡛ PCNA ࡛ࡡ┞பష⏕࡞ Pol31 ᐞࡊ࡙࠷ࡾྊ⬗ᛮ㧏࠷࡛⩻࠻ࡼࡿࡾࠊ ࡆࡡ࡞ࠉPol31 ࡡࣃࢹ࣓࣌ࣞࢡ࡚࠵ࡾ p50 ࡢ Werner ು⩄ⅴᅄ㐿ఎᏄ⏐∸ WRNࠉ࠽ࡻ ࡦWRN ࡛┞பష⏕ࡌࡾࢰࣤࣂࢠ㈹ WRNIP1 ࡛⤎ྙࡌࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾࠊWRN ࡞ࡗ ࠷࡙ࡢ (i) C ᮆ❻ഁ࡚ p50 ࡞┞பష⏕ࡌࡾࡆ࡛ࠉ(ii) ࣃࢹᇰ㣬⣵⬂࡞ p50 ࡛ WRN ࡡ C ᮆ❻ ࢅ㧏Ⓠ⌟ࡈࡎࡾࡆ࡛࡚ p50 p125 ࢅఔࡖ࡙⣵⬂㈹࠾ࡼᰶ࡛ᑻᅹࢅንࡈࡎࡾࡆ࡛ 偆Szekely et al., 2000)ࠉ(iii) ム㥺⟮හ࡚ฝⰾ㓕ザ Pol δ ࡡὩᛮ WRN ῟ຊ࡞ࡻࡽ୕᪴ࡌࡾࡆ ࡛ (Kamath-loeb et al., 2000) ずฝࡈࡿ࡙࠷ࡾࠊୌ᪁ࠉWRNIP1 ࡞ࡗ࠷࡙ࡢム㥺⟮හ࡚ Pol δ ࡞├⤎ྙࡊࠉPol δ ࡡ DNA ྙᠺ㛜╂㢎ᗐࢅ୕᪴ࡈࡎࡾࡆ࡛♟ࡈࡿ࡙࠷ࡾ (Tsurimoto et al., 2005)ࠊ
iii) Third subunit (Pol32)
➠3 ࡡࢦࣇࣗࢼࢴࢹࡢ POL32 ࡞ࢤ࣭ࢺࡈࡿ࡙࠷ࡾࠊPOL32 ࡡ࣓࣌ࣞࢡ㛣࡞࠽࠷࡙ࡢ࣐ ࢿ㓗㒼าࡡ┞⇆ᛮ20偃௧ୖ࡛ࡂࠉ࠵ࡱࡽ⛸㛣࡚┞⇆ᛮずฝࡎ࠷ࠉ࠷ࡍࡿࡵ C ᮆ ❻ഁ࡞ᆵⓏPCNA ⤎ྙ࣓ࢲ࣭ࣆ (PIP box) ࢅ᭯ࡊࠉࡆࡡ㡷ᇡ࡞㝀ࡿࡣ┞⇆ᛮࡢ 30偃⛤ ᗐ࡛ࡾࠊPol32 ࡢฝⰾ㓕ザ Pol δ ࡡ 3 ࡗࡡࢦࣇࣗࢼࢴࢹࡡහࠉ။ୌ PCNA ࡛ࡡ⤎ྙὩᛮ ☔ヾࡈࡿ࡙࠷ࡾࢦࣇࣗࢼࢴࢹ࡚࠵ࡽࠉPCNA ࡞ࡻࡾఘ㛏ಀ㐅ࡢࡆࡡࢦࣇࣗࢼࢴࢹ࡞ኬࡀࡂ౪ Ꮛࡊ࡙࠷ࡾࠊࡱࡒࠉ㓕ザPol δ ⢥⿿ᵾဗࡡ glycerol gradient sedimentation ࡞ࡻࡾゆᯊ࠾ࡼ Pol32 ࡢࣞࢴࢺ≟ࡡᵋ㏸ࢅ࡛ࡖ࡙࠷ࡾ࡛᥆ῼࡈࡿ࡙࠷ࡾ (Johansson et al., 2001)ࠊ
⾪⌟ᆵ࡞㛭ࡊ࡙ࡢฝⰾ㓕ザࡡ 3 ࡗࡡࢦࣇࣗࢼࢴࢹࡡහࠉPOL32 ࡡࡲࡢᚪ㡪㐿ఎᏄ࡚ࡢ
ഭᐐࡡMethyl methanesulfonate (MMS)࡞ᑊࡊ࡙㧏វུᛮࢅ♟ࡊࠉࡆࡡን␏ᰬ࡚ࡢ DNA ഭㄇᑙᛮࡡ✲↓ን␏⋙΅ᑛࡌࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾ (Gerik et al., 1998)ࠊࡱࡒࠉPOL32 Ḗ
ࡢ POL3 ࡡⅤን␏ cdc2-1ࠉPOL31 ࡡⅤን␏ hys2-1 ࡛ྙᠺ⮬ほ࡚࠵ࡾࠊୌ᪁ࠉฦ㓕ザ࡚
ࡢࡆࡡࢦࣇࣗࢼࢴࢹࢅࢤ࣭ࢺࡌࡾcdc27 +ࡢ⣵⬂⑵る࡞ᚪ㡪࡚࠵ࡽ (Hughes et al.,1992)ࠉ々 ྙమࡡᵋ㏸࡛ࡡ㛭㏻ࡢ᪺ࡼ࠾࡚ࡢ࠷ࠉࢦࣇࣗࢼࢴࢹࡡᩐ࡛⇆ᵕ࡞⛸㛣࡚ࡡ⾪⌟ᆵࡡᕣ ␏ヾࡴࡼࡿࡾࠊ ࡆࡿࡱ࡚ࡡゆᯊ࠾ࡼ POL32 㐿ఎᏄࡡᙲࡡୌࡗ࡛ࡊ࡙ࠉPol ζ ࡞౪Ꮛࡊࡒഭࡽ㉲࠻ DNA ྙᠺࡡᐞ᪺ࡼ࠾࡞ࡖ࡙࠷ࡾࠊ々⿿ࣆ࢚࣭ࢠഭ㒂నࢅࡵࡗ㗢ᆵ㙈ࢅ㏳㐛ࡌ ࡾ㝷ࠉPol δ ࡢഭ㒂నࢅ㗢ᆵ࡛ࡊ࡙ຝ⋙Ⓩ࡞ DNA ྙᠺࢅ⾔࠹ࡆ࡛࡚ࡀ࠷ࡒࡴࠉ々⿿ࣆ ࢚࣭ࢠࡢೳでࡌࡾࠊࡆࡡࡻ࠹ࠉ⣵⬂࡞࡛ࡖ࡙༱ᶭⓏDNA ྙᠺࡡೳでࢅᅂ㑂ࡌࡾࡒࡴ ࡞RAD6 ࡞౪Ꮛࡊࡒ PCNA ࡡࣗࣄ࢞ࢲࣤࢅఔ࠹ Post Replication Repair (PRR) ⤊㊨ࡡὩᛮ ㉫ࡆࡽࠉᖼࡗ࠾ࡡ⤊㊨࡞ࡻࡖ࡙々⿿ࣆ࢚࣭ࢠࡡ㐅⾔ࡡ්㛜ಀࡈࡿࡾࡆ࡛▩ࡼࡿ࡙ ࠷ࡾ (Hoege et al., 2002)ࠊࡆࡡ⤊㊨ࡡ࠹ࡔࠉPol ζ (Rev3/Rev7) ࡡ㛭ࢂࡾ⤊㊨ࡡഭࡽ㉲࠻ DNA ྙᠺࡢ✲↓ን␏ࡡⅴᅄࡡୌࡗ࡚࠵ࡾࠊ๑㏑ࡡ࡛࠽ࡽ pol32 ◒ቪᰬ࡞࠽࠷࡙ࡢ DNA ഭㄇⓆᛮࡡ✲↓ን␏⋙ࡡ΅ᑛずࡼࡿࡾࠉࡆࡡ⌟㇗࡞ఔ࠹⣰አ⥲វུᛮࡡஸ㐅ࡢPol ζ ࡡ഼ࡂ⤊㊨࡛⇆ࡋ⤊㊨ࡡᶭ⬗Ḗ࡚࠵ࡾࡆ࡛♟ࡈࡿࡒ (Gerik et al., 1998; Hanna et al., 2007)ࠊࡈࡼ࡞ 2004 ᖳ࡞ࠉPol32 ࡡࢺ࣒ࣤゆᯊ࡞࠽࠷࡙ࡆࡡ΅ᑛ C ᮆ❻㒂ࡡ PIP box 㛭ࡊ࡙࠷ࡾࡆ࡛♟ြࡈࡿ࡙࠷ࡒࡆ࡛࠾ࡼࠉ⣰አ⥲↯ᑏࠉࣗࣄ࢞ࢲࣤ PCNA ࢅࡊ ࡒPol δ ࠾ࡼഭࡽ㉲࠻ᆵ DNA ྙᠺ㓕⣪ࡡୌࡗ࡚࠵ࡾ Rev1 ࡡ࣒࣭࣎ࣛࣚࢭஹᥦཬᚺࡡ 㝷࡞Pol32 㛭ࢂࡖ࡙࠷ࡾ࡛࠷࠹࣓ࢸࣜ⩻࠻ࡼࡿ࡙࠷ࡾ (Johansson et al., 2004)ࠊ
ࡱࡒࠉࢺ࣒ࣤゆᯊ࠾ࡼࡢHU វུᛮࡷῺវུᛮࢅ┞ࡌࡾࡒࡴ࡞ࡢ Pol31 ⤎ྙ㒂࡚࠵
ࡾ N ᮆ❻ഁࡡ㡷ᇡᚪこ࡚࠵ࡽࠉࡆ ࡿࡼࡡⷾ ฌ⌦࡞ᑊࡌࡾ⾪⌟ᆵࡡ┞ࡡ ࡒࡴ࡞
Pol31-Pol32 㛣ࡡ┞பష⏕㔔こ࡚࠵ࡾࡆ࡛♟ြࡈࡿ࡙࠷ࡾ (Johansson et al., 2004)ࠊ ࡆࡿࡼࡡ⾪⌟ᆵ࡞ࡗ࠷࡙ࡢ᭩㎾ࡱ࡚⏍ᏕⓏᶭ⬗࡛ࡡ㛭㏻ᛮ᪺ࡼ࠾࡚ࡢ࠾ࡖࡒࠉ 2007 ᖳ࡞ฝⰾ㓕ザ⣵⬂ࢅ⏕࠷ࡒ◂✪࠾ࡼ Pol32 ࡡ N ᮆ❻ഁ㡷ᇡ BIR (Break induced
replication) ࡞ᐞࡊ࡙࠷ࡾࡆ࡛ሒ࿈ࡈࡿࡒ (Lydeard et al., 2007)ࠊBIR ࡢ DNA 々⿿ࡡ㏭୯ ࡚ᮇ㙈ว᩷⏍ࡋࡒ㝷࡞ࠉ┞⇆⤄ࡲᥦ࠻ࢅฺ⏕ࡊ࡙DNA 々⿿ࢅ්㛜ࡌࡾᶭᵋ࡚࠵ࡾࠊBIR ࡢೳでࡊࡒ々⿿ࣆ࢚࣭ࢠࡷቪࡿࡒ々⿿ࣆ࢚࣭ࢠ࡚々⿿ࢅ්㛜ࡈࡎࠉࠔ᭨ࢷ࣒ࣞ⥌ᣚᶭ ᵋࠕ࡛ࡻࡣࡿࡾ⤊㊨࡞࠽࠷࡙ࠉࢷ࣒࣭ࣞࣚࢭࢅࡵࡒ࠷⣵⬂ࡡ▯⦨ࡊࡗࡗ࠵ࡾࢷ࣒ࣞ࡞ ష⏕ࡊࠉࢷ࣒ࣞ㛏ࡡ⥌ᣚ࡞ᐞࡊ࡙࠷ࡾ࡛⩻࠻ࡼࡿ࡙࠷ࡾࠊࡆࡡ◂✪࠾ࡼPol δ ࠉBIR ࡡ㝷ࡡ᩺ぜDNA ྙᠺ࡞࠽ࡄࡾࣈ࣏࣭ࣚఘ㛏ア㝭ࡡิ࡞㛭ࢂࡾࡆ࡛ሒ࿈ࡈࡿࡒࠊ ฝ ⰾ 㓕 ザ ࡞ ࡢ ず ฝ ࡈ ࡿ ࡙ ࠷ ࠷ ࢦ ࣇ ࣗ ࢼ ࢴ ࢹ
iv) Fourth subunit
4 ␊┘ࡡࢦࣇࣗࢼࢴࢹࡢฝⰾ㓕ザ࡞ࡢᏋᅹ☔ヾࡈࡿ࡙࠷࠷ࠉࢦࣇࣗࢼࢴࢹ㛣ࡡ┞ப ష⏕࡛࠷࠹びⅤ࠾ࡼᮇ◂✪ࡡゆᯊࡡ⌦ゆ࡛⩻ᐳ࡞ᙲ❟࡙ࡼࡿࡾ࡛ᛦࢂࡿࡾࡡ࡚ゆㄕࡊ࡙࠽ ࡂࡆ࡛࡞ࡌࡾࠊฦ㓕ザ࡚ࡢࡆࡡࢦࣇࣗࢼࢴࢹࢅࢤ࣭ࢺࡌࡾ cdm1 +㐿ఎᏄࡢᙔิࠉPol31 ࣓࣌ࣞࢡ Cdc1 ࡡῺᗐវུᛮን␏ cdc13-P13 ࡡ Multicopy suppressor ࡛ࡊ࡙ずฝࡈࡿࡒ (MacNeill et al., 1996)ࠊᚃ࡞ฦ㓕ザ࠾ࡼ⢥⿿ࡊࡒ Pol δ ࡐࡿࡱ࡚࡞⇆ᏽࡈࡿ࡙࠷ࡒ 125 kDaࠉ 55 kDaࠉ54 kDa (ࠍPol3ࠉCdc1ࠉCdc27) ࡡࢰࣤࣂࢠ㈹௧አ࡞ 22 kDa ࡡࢰࣤࣂࢠ㈹ࢅྱࢆ ࡚࠷ࡾࡆ࡛ฦ࠾ࡽࠉPol δ ࡡࢦࣇࣗࢼࢴࢹ࡚࠵ࡾࡆ࡛ึ᪺ࡊࡒ (Zuo et al., 1997 )ࠊࡱࡒࠉ ┞பష⏕࡞ࡗ࠷࡙ࡢPol3ࠉPol31 ࣓࣌ࣞࢡ࡞⤎ྙࡌࡾࡆ࡛ࠉ⾪⌟ᆵ࡞㛭ࡊ࡙ࡢࡆࡡ㐿ఎᏄࡡ ◒ቪᰬࡡ DNA ഭᐐ࡞ᑊࡌࡾវུᛮࡢ㔕⏍ᰬ࡛࡛ࢆንࢂࡼ࠷ࡆ࡛▩ࡼࡿ࡙࠷ࡾ (Reynolds et al., 1998)ࠊ ୌ᪁ࠉ⢥⿿ࡈࡿࡒࣃࢹࡡ࣓࣌ࣞࢡࡡp12 ࡢฦ㓕ザ࡛⇆ᵕ࡞ Pol3ࠉPol31 ࣓࣌ࣞࢡ࡞⤎ྙ ࡌࡾࠊࡱࡒࠉム㥺⟮හ࡚ࡢ༟≺࡚PCNA ࡛ࡵ┞பష⏕ࡊࠉࡆࡡ┞பష⏕ Pol δ ࡡὩᛮࠉཬ ᚺࡡᣚ⤾ᛮ࡞ୌᏽࡡᐞ࠵ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Li et al., 2006)ࠊࡊ࠾ࡊࡼࠉࡆࡡ ┞பష⏕ࢅḖࡂን␏p12 ࡞ࡻࡖ࡙ᵋᠺࡈࡿࡾ Pol δ ࡢ PCNA ࡞ࡻࡖ࡙Ὡᛮࡈࡿࡾࡆ࡛ྊ ⬗࡚࠵ࡾࡡ࡞ᑊࡊ࡙ࠉp12 ࡐࡡࡵࡡࢅḖ࠷ࡒ Pol δ ࡢ PCNA ࡞Ὡᛮࡈࡿ࠷ࡆ࡛࠾ࡼ PCNA
࡞ࡻࡾὩᛮࡡ p12 ࡡᐞࡢࡆࡡ┞பష⏕࡞Ᏸධ࡞౪Ꮛࡊࡒࡵࡡ࡚ࡢࡂࠉౚ࠻ࡣ
ࡈࡿ࡙࠷ࡾࠊࡈࡼ࡞᭩㎾࡚ࡢ⣰አ⥲ࠉMMSࠉHUࠉAphidicolin ฌ⌦࡞ࡻࡽ DNA 々⿿ࢪࢹࣝ ࢪࢅࣃࢹᇰ㣬⣵⬂࡞࠻ࡾ࡛ࣈࣞࢷࢮ࣭࣑౪ᏋⓏ࡞p12 ฦゆࡈࡿࡾࡆ࡛ሒ࿈ࡈࡿࡒࠊ ࡆࡡฦゆࡢS ࡡ DNA ഭᚺ➽࡞㛭ࡌࡾ ATR ࢅḖࡊࡒ⣵⬂࡚ࡢ㉫ࡆࡼ࠷ࡆ࡛࠾ࡼࠉ DNA 々⿿ࢅโᚒࡌࡾഭᚺ➽ࡡୌࡗ࡞ p12 ᐞࡊ࡙࠷ࡾࡆ࡛♟ြࡈࡿ࡙࠷ࡾ (Zhang et al., 2007)ࠊ Subunits
Species Large subunit Second subunit Third subunit Fourth subunit
S. cerevisiae POL3 (Pol3)
POL31/HYS2/SDP5
(Pol31) POL32 (Pol32)
S. pombe pol3 +(Pol3) cdc1+ (Cdc1) cdc27+(Cdc27) cdm1+(Cdm1)
H. sapiens POLD1 (p125) POLD2 (p50) POLD3 (p68) POLD4 (p12)
Table 1 Subunits of Pol δ Names of gene encoding Pol δ subunits in S. cerevisiae, S. pombe and H. sapiens are summarized. Names of protein are given in parentheses. (red: lethal, violet: hyper-sensitive to DNA damage, blue: none-sensitive to DNA damage, black: N. D.)
Fig. 5 Schematic models of subunit interactions In S. cerevisiae, Pol δ is a trimeric enzyme consisting of 125, 55 and 40 kDa polypeptides, corresponding to the catalytic subunit Pol3, Pol31 and Pol32 (panel a). In S. pombe (panel b) and H. sapiens (panel c), the complex contains a fourth small subunit referred to as Cdm1 and p12, respectively (Table 1).
➠
4 ⟿ ᮇ◂✪ࡡ┘Ⓩ
๑㏑ࡊࡒ࡛࠽ࡽࠉDNA 々⿿ࣆ࢚࣭ࢠࡡೳで࡞ࡻࡾ々⿿ࡡ␏ᖏࡢࢆࡡ⑄ᝀ࡛ᐠ࡞ 㛭㏻ࡊ࡙࠷ࡾࠊࡱࡒࠉPol δ ࡢ DNA 々⿿࡚୯ᚨⓏᙲࢅᢰ࠹㓕⣪࡚࠵ࡽࠉ々⿿ೳでࡊࡒ ᚃ࡚ࠉ々⿿්㛜ࡈࡿࡾ࡞ࡵ㔔こᶭ⬗ࢅᢰ࠹ࡆ࡛♟ြࡈࡿ࡙࠷ࡾࠊࡊࡒࡖ࡙ࠉPol δ ࢅ⌦ゆࡌࡾࡆ࡛ࡢࠉࢣࢿ࣑ࡡᏭᏽ⥌ᣚᶭᵋࡡ⌦ゆ࡞ࡗࡽࠉࡱࡒࠉࡐࡡᶭ⬗Ḗ࡞ࡻࡾ⑄ ᝀࡡⓆࡡᶭᗆࢅ⌦ゆࡌࡾࡒࡴ࡞ᙲ❟࡙ࡼࡿࡾ࡛⩻࠻ࡼࡿࡾࠊ Pol31 ࡢ Pol δ ࡡࢦࣇࣗࢼࢴࢹ࡚࠵ࡽࠉฝⰾ㓕ザ࡚ࡢ⣵⬂⑵る࡞ᚪ㡪ᶭ⬗ࢅᯕࡒࡊ࡙࠷ ࡾࠊPol31 ࡡ⏍ᏕⓏὩᛮ࡞㛭ࡊ࡙ࡢ Pol3 ࡛ Pol32 ࡛⤎ྙࡊࠉ୦ࢦࣇࣗࢼࢴࢹࢅ㏻⤎ࡊ࡙ ࠷ࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾࠉࡆࡿࡱ࡚Pol31 ࡡᶭ⬗ࡡリ⣵࡞ࡗ࠷࡙ࡢ࡛ࢆゆᯊࡈࡿ࡙ࡆ ࠾ࡖࡒࠊࡐࡆ࡚Pol31 ࡡᶭ⬗ࢅゆᯊࡌࡾࡒࡴ࡞ࠉPol δ 々ྙమࡡ୯࡚ࡡࢦࣇࣗࢼࢴࢹ㛣ࡡ ┞பష⏕ࢺ࣒ࣤࡡ≁ᏽࢅ┘ᣞࡊࠉゆᯊࢅ⾔ࡖࡒࠊ➠
3 ❮ ⤎ᯕ
➠ 1 ⟿ Pol δ 々 ྙ మ ᙟ ᠺ ࡞ 㛭 ࡌ ࡾ ࢺ ࣒ ࣤ ࡡ ᥀ ⣬
Pol31 ࡡ࣓࣌ࣞࢡ࡞࠽࠷࡙ࡢࡐࡡ࣐ࢿ㓗㒼าࣃࢹ࠾ࡼ㓕ザࡱ࡚㧏ᗐ࡞ಕᏋࡈࡿࡒ 10 ࡡ㡷ᇡᏋᅹࡌࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Fig. 6) (Reynolds et al., 1999)ࠊࡊ࠾ࡊࠉࡆࡿࡼࡡ 㡷ᇡࢅᡥ࠾ࡽ࡛ࡊࡒPol31 ࡡᶭ⬗ࡡゆᯊࡢ⾔ࢂࡿ࡙࠽ࡼࡍࠉ⣵⬂⑵る࡞ᚪ㡪ᶭ⬗࡞ࡗ࠷ ࡙ࡵࡐࡡリ⣵࡞ࡗ࠷࡙ࡡሒ࿈ࡢ࠷ࠊ
Fig. 6 Protein alignment of Pol31/PolD2 proteins from ten eukaryotic species (Reynolds et al., 1999) Based on protein sequence comparisons across several eukaryotic species, ten conserved regions have been identified in Pol31.
ࡐࡆ࡚ࡱࡍࠉ┞பష⏕ࢺ࣒ࣤࡡᡥ࠾ࡽࢅᚋࡾࡒࡴࠉ2xHA ࢰࢡࡗࡀࡡ N ᮆ❻ഁࠉC ᮆ ❻ഁࡡ⣑༖ฦࡡ㡷ᇡࢅࢤ࣭ࢺࡌࡾࡌࡾࣈࣚࢪ࣐ࢺࢅ⏕࠷࡙ฝⰾ㓕ザ⣵⬂හ࡚Ⓠ⌟ࡈࡎࠉඞ ỷ㜾Ἢ࡞ࡻࡾභỷ㜾ᐁ㥺ࢅ⾔ࡖࡒࠊࢣࢿ࣑୕ࡡ POL31 㐿ఎᏄࡢ㟸◒ቪ࡚ࠉࢣࢿ࣑୕ࡡ POL3 ࡛ POL32 㐿ఎᏄ࡞ࡢ 3xFLAG ࢰࢡࢅ C ᮆ❻ഁ࡞ຊࡊࡒ㓕ザࢅࡐࡿࡑࡿష⿿ࡊ偆Fig. 7A, panel a假ࠉࡐࡡᰬ࡞ᑊࡊ࡙ pol31 㐿ఎᏄࢅ GAL1 ࣈ࣓࣭ࣞࢰ࣭ୖὮ࡞⦽࠷ࡓ DNA ࢅྱࡳ ࣈࣚࢪ࣐ࢺࢅᑙථࡊࡒࠊࡆࡡࣈࣚࢪ࣐ࢺᑙථࡈࡿࡒ㓕ザ࡚ࡢᇰᆀ࡞ galactose ࢅ῟ຊࡌࡾ
࡛ GAL1 ࣈ࣓࣭ࣞࢰ࣭࠾ࡼࡡ㐿ఎᏄ㌷ಀ㐅ࡈࡿࠉglucose ῟ຊ࡞ࡢ㌷ᢒโࡈࡿࠉ ⤎ᯕⓏ࡞┘Ⓩ࡛ࡌࡾࢰࣤࣂࢠ㈹ࡡⓆ⌟ࡡ on/off ࢅࢤࣤࢹ࣭ࣞࣜࡌࡾࡆ࡛࡚ࡀࡾ (Fig. 7A, panel b )ࠊࡆࡡᰬࢅᑊᩐ⑵るࡱ࡚ᇰ㣬ᚃࠉ⤂⃨ᗐ 2偃࡞ࡾࡻ࠹࡞ galactose ࢅ῟ຊࡊࡒࠊ ࡆࡡ⣵⬂ࡡฝᾦࢅ⏕࠷࡙ᢘ HA ᢘమ࡚ඞỷ㜾ࡊࠉLysis buffer ࡚ὑὯࡊࡒỷ㜾∸ࢅ㞹ポ ὃິ࡚ฦ㞫ᚃࠉWestern blot Ἢ࡚ Pol3-FLAGࠉPol32-FLAG ࢅ᳠ฝࡊࡒࠊ
ࡐࡡ⤎ᯕࠉPol3 ࡢࠉPol31 ࡡ N ᮆ❻ഁ㡷ᇡ I-V ࢅྱࡳ 1-275 aa ᩷∞ࠉC ᮆ❻ഁ㡷ᇡ VI-X ࢅྱࡳ279-487 aa ᩷∞ࡡࡔࡼࡡ᩷∞࡛ࡵභỷ㜾ࡊࡒࠊୌ᪁ࠉPol32 ࡢ 1-275 aa ᩷∞࡞࡛ࡢ භỷ㜾ࡊࡒࡵࡡࡡࠉ279-487 aa ᩷∞࡛ࡢභỷ㜾ࡊ࠾ࡖࡒ (Fig. 7B)ࠊ
Pol3 ࡡ Pol31 ࡞ᑊࡌࡾ⤎ྙࢺ࣒ࣤࡢ C ᮆ❻㒂ࡡ C4 Zn-finger 㡷ᇡ࡚࠵ࡽࠉࡆࡡ㡷ᇡࡡ C1056ࠉC1059ࠉC1069ࠉC1074 ࡡⅤን␏࡚┞பష⏕㢟ⴥ࡞΅ᘽࡌࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ ࡾ (Sanchez Garcia et al., 2004)ࠊࡊࡒࡖ࡙ࠉPol3 ࡡ Pol31 ࡞ᑊࡌࡾ┞பష⏕ࡢ㝀ࡼࡿࡒ㡷 ᇡ࡚⥌ᣚࡈࡿ࡙࠷ࡾ࡛⩻࠻ࡼࡿࡾࠊୌ᪁ࠉPol31 ࡡ 275 aa ㎾ࡢಕᏋ㡷ᇡ࡚ࡢ࠷ࡆ࡛࠾ࡼ Pol31 ഁࡡ┞பష⏕Ⅴࡢ 1 ḗᵋ㏸୕ࠉ㞫ࡿࡒⅤ࡞々ᩐᏋᅹࡊࠉPol3 Zn-finger ࡞ᑊࡊ࡙ Pol31 ࡢ々ᩐࡡ㒂న࡚⤎ྙࡊ࡙࠷ࡾྊ⬗ᛮ㧏࠷࡛⩻࠻ࡼࡿࡾ 偆Fig. 8假ࠊ
Fig. 7 The N-terminal region of Pol31 is sufficient to constitute the Pol δ complex between Pol3 and Pol32. A, Immunoblotting of Pol δ subunits. (a) Extracts were prepared from cells expressing Pol3-FLAG, Pol31-FLAG or Pol32-FLAG and fractionated by SDS-PAGE followed by immunoblotting with an anti-FLAG monoclonal antibody. (b) Extracts were prepared from cells carrying a multicopy plasmid encoding HA-Pol31 protein, which had been cultured in the medium containing galactose for 12 h at 30|C B, Immunoprecipitaton of Pol δ subunits. The Pol32-FLAG and Pol3-FLAG cells which were transformed with pKT10L-P31, pKT10L-p31-N5 (1-275 a.a.), pKT10L-C2 (279-487 a.a.), or pKT10L (empty) plasmids, were incubated for 12 hr in the presence of galactose, and then the cells were lysed by the glass bead method. HA-tagged proteins were immunoprecipitated with anti-HA antibody-conjugated agarose beads as described under Experimental Procedures. Immunoprecipitates were fractionated by SDS-PAGE and analyzed by immunoblotting with an anti-FLAG monoclonal antibody as well as with anti-HA antibody. I.B., immunoblotting; I.P., immunoprecipitation.
Fig. 8 Summary of subunit interactions expected in this experiment. The N-terminal 1-275 aa region of Pol31 could coimmunoprecipitate both Pol3 and Pol32 whereas the C-terminal 279-487 aa region only coimmunoprecipitated Pol3. Thus, it seems likely that Pol31 has two regions for interacting with Pol3 but only one for interacting with Pol32.
➠ 2 ⟿ ⣵ ⬂ ⑵ る ⬗ ࢅ ࠻ ࡾ 㡷 ᇡ ࡡ ᥀ ⣬
N ᮆ❻ഁࡡ᩷∞ࡢ Pol3 ࡛ Pol32 ࡡ୦᪁࡞⤎ྙࡊࠉ々ྙమࡡᙟᠺྊ⬗࡚࠵ࡾࡆ࡛♟ြ ࡈࡿࡒࠊࡐࡆ࡚ࠉඞỷ㜾Ἢ࡚⏕࠷ࡒGAL1 ࣈ࣓࣭ࣞࢰ࣭࡚โᚒ࡚ࡀࡾࣈࣚࢪ࣐ࢺⓆ⌟⣌ࢅ ⏕࠷࡙ࠉPol3 ࡡ᩷∞ࢅⓆ⌟ࡌࡾ⣵⬂ࡡ⑵る⬗ࢅㄢࡒࠊ
Fig. 9 Scheme of evaluation of deleted Pol31 protein for viability. The phenotype conferred by a pol31 deleted allele carried by the LEU2 plasmid is revealed after loss of the wild type
POL31 gene on the URA3 plasmid following
5-FOA selection, because cell viability is now viability is now dependent on the mutated
pol31 allele.
ฝⰾ㓕ザ POL31 ࡢᚪ㡪㐿ఎᏄ࡚࠵ࡾࡒࡴ࡞ḗࡡࡻ࠹ࢹࣛࢴࢠࢅ⏕࠷࡙ን␏ࢰࣤࣂࢠ
㈹ࡡᶭ⬗ࢅῼᏽࡊࡒࠊࡆࡡ᪁Ἢ࡚ࡢᵾⓏ㐿ఎᏄࢅ◒ቪࡌࡾ๑࡞ࠉ㔕⏍ᆵࡡ POL31 ࡞ URA3 ࣏࣭࣭࢜ࢅ㏻⤎ࡊࡒ࡞ࣈࣚࢪ࣐ࢺࢅᑙථࡊࠉࡐࡡᚃࠉ⣵⬂ࡡPOL31 㐿ఎᏄࡡ◒ቪࢅ⾔࠹ࠊ ࡈࡼ࡞ࡐࡡᰬ࡞┘Ⓩࡡን␏ࢅࡵࡗpol31 ࢅ㏻⤎ࡊࡒࣈࣚࢪ࣐ࢺࢅᑙථࡊࡒ偆Fig. 9, left panel假ࠊ 5-fluoroorotic acid (5-FOA) ࡐࡡࡵࡡࡢスᛮࢅᣚࡒ࠷ URA3 㐿ఎᏄࢅⓆ⌟ࡌࡾ㓕ザ࡞ཱི ࡽ㎰ࡱࡿࡾ࡛ URA3 㐿ఎᏄ⏐∸ (Orotidine 5’-phosphate-decarboxylase) ࡡよ፳ష⏕࡞ࡻࡽ 5-fluoro-orotidine monophosphateࠉࡈࡼ࡞ࡢ 5’-fluoro-uridine monophosphate (5-FUMP) ࡞ ㅨࡈࡿࡾࠊ5-FUMP ࡢࢲ࣐ࢩࣜ㓗 (DNA ࡡᵋᠺᠺฦ) ࡡྙᠺ㜴ᐐ∸㈹࡚࠵ࡾࡒࡴࠉ⣵⬂࡞࡛ ࡖ࡙᭯ᐐ࡛ࡾࠊࡐࡡࡒࡴࠉࡆࡡ᭯ᐐ∸㈹ࡡྙᠺࢅ㜭ࡃ࡞ࡢURA3 㐿ఎᏄ⏐∸ࡡᶭ⬗Ḗ ࡌࡾᚪこ࠵ࡾࠊࡊࡒࡖ࡙ 5-FOA ࢅྱ᭯ࡌࡾᇰᆀ୕࡚ࡢ URA3 ࡛㏻⤎ࡊࡒ㔕⏍ᆵ㐿ఎᏄ ࢅࡖࡒ⣵⬂㐽ᢝࡈࡿࡾࡆ࡛࡞ࡾࠊ5-FOA ྱ᭯ᇰᆀ୕࡚⑵る࡚ࡀࡾᰬࡢᑙථࡊࡒን␏ ᆵ㐿ఎᏄ࡞౪Ꮛࡊ࡙⏍Ꮛࡊ࡙࠷ࡾࡆ࡛࡞ࡾ 偆Fig. 9, right panel假ࠊࡌࢂࡔࠉ5-FOA ྱ᭯ ᇰᆀ࡚ࢤࣞࢼ࣭ࢅᙟᠺࡊࡒሔྙࠔን␏Pol31 ࡞ࡻࡖ࡙⣵⬂⑵る⬗ྊ⬗࡚࠵ࡾࠕ࡛ึ᩷ࡊࡒࠊ
୕エࡡࡻ࠹᪁Ἢ࡞ࡻࡽN ᮆ❻ഁ Pol31 ᩷∞࡞ࡻࡖ࡙⣵⬂⑵る⬗ࡡビ౮ࢅ⾔ࡖࡒࠊࡐࡡ ⤎ᯕࠉḖን␏మ࡚ࡢ᭩▯࡚1-113 aa ᩷∞ (I-II ࡡ༖ฦ假 ࢅ㐛Ⓠ⌟ࡊࡒሔྙࠉ⑵るྊ⬗ ࡚࠵ࡖࡒࡡ࡞ᑊࡊ࡙ࠉ1-96 aa 偆I假 ᩷∞ࢅ㐛Ⓠ⌟ࡊࡒ⣵⬂ࡢ⑵る࡚ࡀ࠾ࡖࡒࠊࡱࡒࠉ N ᮆࡡ 113 aa ࡻࡽ㛏࠷᩷∞ࠉ1-455 aa (I-IX)ࠉ1-363 aa (I-VII)ࠉ1-275 aa (I-V) (data not shown)ࠉ1-229 aa (I-IV)ࠉ1-192 aa (I-III)ࠉ1-143 aa (I-II)ࠉ 1-129 aa (I-II ࡡ༖ฦ) (Fig. 10A) ࢅ
㐛Ⓠ⌟ࡊࡒ⣵⬂ࡢ⑵るྊ⬗ࡓࡖࡒࠊࡒࡓࡊࠉ1-417 aa ࢅ㐛Ⓠ⌟ᰬࡊࡒ⣵⬂ࡢ⑵る࡚ࡀ ࠾ࡖࡒ (data not shown)ࠊ
ḗ࡞ࡆࡿࡼࡡ⣵⬂⑵る⬗ࡡ᭯↋Ⓠ⌟㔖࡞౪Ꮛࡊࡒࡵࡡ࡚࠷ࡆ࡛ࢅ☔࠾ࡴࡾࡒࡴ࡞ࠉ ⇆ᵕࡡ᮪௲࡚㐛Ⓠ⌟ࡈࡎࡒ⣵⬂ࡡฝᾦࢅ⏕࠷࡙Pol31 ᩷∞ࡡ㔖ࢅ Western blot Ἢ࡞ࡻࡽ セ㍉ࡊࡒ(Fig. 10B)ࠊࡐࡡ⤎ᯕࠉ1-113 aa ࡛ 1-96 aa ࡡ㛣࡞ࡢⓆ⌟㔖࡞㢟ⴥᕣࡢ↋ࡂࠉ⣵⬂ ⑵る⬗ࡡ᭯↋ࡢ㡷ᇡࡡḖ࡞౪Ꮛࡊࡒࡵࡡ࡚࠵ࡾࡆ࡛♟ြࡈࡿࡒࠊ ࡱࡒࠉ㔕⏍ᆵ POL31 ࡡࣈࣚࢪ࣐ࢺ 偆URA3 ࣏࣭࣭࢜假 ࡡᑙථࢅ⤊⏜ࡊ࡙⣵⬂ᰬࢅష⿿ ࡊ࡙࠷ࡾࡒࡴࠉᏰධ࡞ࡆࡡࣈࣚࢪ࣐ࢺࢅḖࡊ࡙࠷ࡾ࠾࠹࠾࡞ࡗ࠷࡙☔ヾࡊࡒ࡛ࡆࢀࠉ 5-FOA ྱ᭯ಶᆀ୕࡚ࢤࣞࢼ࣭ࢅషࡖࡒᰬࡢࣚࢨࣜḖᇰᆀ࡚ࡢࢤࣞࢼ࣭ࢅషࡼ࠾ࡖࡒ ࡆ࡛࠾ࡼࠉ㔕⏍ᆵ㐿ఎᏄࡡΊᅹࡡྊ⬗ᛮࡢྫྷᏽࡈࡿࡒ 偆Fig. 10C假ࠊ ㏳ᖏࠉGAL1 ࣈ࣓࣭ࣞࢰ࣭࡞ࡻࡾ㌷ㄢ⟿ࢅུࡄࡾࡻ࠹㐿ఎᏄ࡞ᑊࡊ࡙ glucose ࢅ῟ຊ ࡊࡒሔྙࠉࡐࡡ㐿ఎᏄḖ࡛⇆➴ࡡ⾪⌟ᆵࢅ♟ࡌࡆ࡛ኣ࠷ࠊࡊ࠾ࡊࠉධ㛏Pol31 ࢅⓆ⌟ࡌ ࡾࣈࣚࢪ࣐ࢺࢅᑙථࡊࡒ⣵⬂ࡢࠉglucose ῟ຊ (㌷ᢒโ) ࡚ࡵ⑵るྊ⬗࡚࠵ࡖࡒࠊ glucose ῟ຊ࡞ࡢࢰࣤࣂࢠ㈹ࡵ᳠ฝ࡚ࡀ࠾ࡖࡒࡒࡴࠉࡆࡡᰬࡡࢤࣞࢼ࣭ᙟᠺ࡞ၡよ ࡖࡒࠉRT-PCR ࢅ⾔ࡖࡒ࡛ࡆࢀࠉ㌷ࡢࢂࡍ࠾ࡼ㉫ࡆࡖ࡙࠷ࡾࡆ࡛ึ᪺ࡊࠉධ㛏 Pol31 ࢰࣤࣂࢠ㈹ࡡ᚜㔖ࡡⓆ⌟࡚ࡵ⣵⬂⑵る࡞ࡢ༎ฦ࡚࠵ࡾࡆ࡛♟ြࡈࡿࡒ (Fig. 11D)ࠊ
Fig. 10 The N-terminal 113 aa fragment of Pol31 is sufficient for viability. A, Evaluation of N-terminal fragments of Pol31 for viability. (b). B, Expression of various N-terminal fragments of Pol31. Extracts were prepared from the cells expressing N-terminal fragments of Pol31 as described under Experimental Procedures, and then fractionated by SDS-PAGE. HA-tagged Pol31 were detected by immunoblotting with an anti-HA antibody. Positions of the molecular mass standard (kDa) are shown on the left of the figure and the asterisk indicates position of non-specific band. C, Confirmation that the N-terminal 113 aa fragment of Pol31 is sufficient for viability. Cells supposedly containing the indicated plasmids shown in panel a, were streaked on SC-Ura plates containing Leu, raffinose and galactose (panel b), SC-Leu plates containing raffinose and galactose (panel c), or SC-Leu plates containing raffinose, and glucose (panel d), and photographed after 5 days incubation at 25|C. To isolate cells containing full length or 1-113 POL31 on the pKT10L plasmid (LEU2 marker) without full length POL31 on pRS316 (URA3 marker), we selected cells containing full length POL31 on pRS316 that could grow on plates containing 5-FOA and used them in this experiment. D, The comparison of POL31 mRNA in the cells grown in medium containing galactoce or glucose. RNA: RNA template, DNA: cDNA template
ḗ࡞C ᮆ❻ഁ࡞ࡗ࠷࡙⇆ᵕࡡゆᯊࢅ⾔ࡖࡒࠊࡌࡾ࡛ N ᮆ❻ഁ 1-107 aa (㡷ᇡ I ࡛ II ࡡୌ㒂) ࢅḖࡊࡒ108-487 aa ࢅⓆ⌟ࡌࡾ⣵⬂ࡢ⑵る࡚ࡀ࠾ࡖࡒࠊࡆࡡࠉ279-487 aaࠉ364-487 aaࠉ 417-487 aa ᩷∞ࢅⓆ⌟ࡌࡾ⣵⬂ࡵ⑵る࡚ࡀ࠾ࡖࡒ (Fig. 11A)ࠊ N ᮆ❻ഁࡡሔྙ࡛⇆ᵕ࡞Ⓠ⌟㔖ࡡセ㍉ࢅ⾔ࡖࡒ࡛ࡆࢀ 108-487 aa ࡞ࡗ࠷࡙ධ㛏࡛࡛ࢆ ንࢂࡼ࠷Ⓠ⌟㔖࡚࠵ࡖࡒࡆ࡛࠾ࡼࠉࡆࡿࡼࡡᰬࡡ⣵⬂⑵る⬗ࡡḖࡢ N ᮆ❻ഁ㡷ᇡࡡ Ḗ࡞ࡻࡾࡆ࡛♟ြࡈࡿࡒ 偆Fig. 11B假ࠊ
࠽ࠉᅒ୯allow head ࡚♟ࡈࡿ࡙࠷ࡾࣁࣤࢺࡢ K379 ࡚ SUMO ಞ㣥ࡈࡿࡒ Pol31 ࡚࠵ࡾ 偆๑ ⏛༡⒬ㄵᩝ假ࠊ⌟ᅹࡡ࡛ࡆࢀࠉࡆࡡ㒂నࡡ࣐ࢪࢬࣤࢪን␏ DNA ഭᐐ࡞ᑊࡊ࡙វུᛮࢅ⑵ ຊࡈࡎࡾ࡛࠷࠹⤎ᯕࡢᚋࡼࡿ࡙࠽ࡼࡍࠉࡆࡡಞ㣥ណ⩇ࡢ᪺࡚࠵ࡾࠊ
Fig. 11 Various N-terminally truncated Pol31 derivatives do not support cell viability A, Viability. Cells carrying plasmids indicated in panel (a), were streaked onto SC-Ura plates containing Leu and raffinose, SC-Leu plates containing raffinose, galactose, and 5-FOA (panel c) or SC-Leu plates containing raffinose, glucose, and 5-FOA (panel d), and photographed after 5 days incubation at 25|C. B, Expression of various N-terminally truncated HA-Pol31 derivatives. Extracts were prepared from the cells expressing N-terminally truncated Pol31 as described under Experimental Procedures, and then fractionated by SDS-PAGE. HA-tagged Pol31 was detected by immunoblotting with an anti-HA antibody. Positions of the molecular mass standards (kDa) are shown on the left of the figure and the asterisk indicates the position of non-specific band. Upper bands (arrow heads) seem to be modified truncated Pol31
Fig. 12 Summary of the viability.
Relationships between trancated Pol31 proteins and the viability of the cells overexpressing the fragment are summarized.
➠ 3 ⟿ 1-113 aa ᩷ ∞ 㐛 Ⓠ ⌟ ᰬ ࡡ ⾪ ⌟ᆵ 1-113 aa ᩷∞㐛Ⓠ⌟ᰬ࡚⣵⬂⑵る⬗ࡢ⥌ᣚࡈࡿ࡙࠷ࡒࠉPol δ ࡢ DNA 々⿿࡞ᚪ㡪࡚࠵ ࡾࡒࡴࠉን␏ᰬ࡚⣵⬂࿔ࡡ␏ᖏびᐳࡈࡿࡾྊ⬗ᛮ⩻࠻ࡼࡿࡾࠊࡐࡆ࡚ࠉ5-FOA ྱ᭯ ᇰᆀ࡚㐽ᢝࡈࡿࡒ N ᮆ❻ഁࡡ㐛Ⓠ⌟ᰬ࡞ࡗ࠷࡙⣵⬂࿔୕ࡡᣪິࢅㄢࡾࡒࡴ࡞ࣆ࣭ࣞ ࢦࢹ࣒ࢹ࣭ࣛ࡞ࡻࡾゆᯊࢅ⾔ࡖࡒࠊࡌࡾ࡛ࠉ1-113 aa ᩷∞Ⓠ⌟ᰬࡢ 2偃 galactose ῟ຊࡢ 㔕⏍ᆵ࡛ንࢂࡼ࠷ᣪິࢅずࡎࡒ (Fig. 13A)ࠊ ୌ᪁ࠉ2偃 glucose ῟ຊ࡞ࡢ 1-113 aa ᩷∞Ⓠ⌟ᰬࡢ S ᚃ࠾ࡼ G2/M ࡞✒ࡌࡾࡻ ࠹࡞ࡽࠉDumpbell ≟ࡡ␏ᖏᙟឺ⣵⬂ࡡྙⴥࡊࡂ⑵ຊࡊ࡙࠷ࡒࠊࡊࡒࡖ࡙ࠉ㌷ ㄇᑙࠉᢒโࡡᇰ㣬᮪௲࡞ࡻࡖ࡙⣵⬂࿔ࡡᣪິንࡌࡾࡆ࡛࠾ࡼࠉࡆࡡᰬPol31 ᩷∞࡞ ౪Ꮛࡊࡒ⣵⬂⑵るࢅ⾔ࡖ࡙࠷ࡾࡆ්࡛☔ヾࡈࡿࡒࠊࡱࡒࠉࡆࡡᰬࡡ㌷ᢒโࡡ⣵⬂࿔ ࡡᣪິ௧๑࡞ሒ࿈ࡈࡿࡒ hys2-1 ᰬࡡ㟸スᐖῺᗐ࡚ࡡᣪິ࡞Ⰳࡂజ࡙࠷ࡾࡆ࡛࠾ࡼ S ᚃ࠾ࡼG2/M ࡡ✒ࡢ Pol31 ࡡᶭ⬗Ḗ࡚࠵ࡾࡵࡡ࡛⩻࠻ࡼࡿࡒ (Fig. 13B) (Sugimoto et al., 1995)ࠊ
ḗ࡞ࠉDNA 々⿿㜴ᐐࡷ DNA ഭᐐ࡞ᑊࡌࡾវུᛮ࡛ῺᗐវུᛮࢅㄢࡒࠊHU ࡢ๑㏑ ࡊࡒ࡛࠽ࡽ RNR 㜴ᐐ࡚࠵ࡽࠉࡆࡡ∸㈹࡞ࡻࡖ࡙ DNA ྙᠺࡡᮞᩩ࡚࠵ࡾ dNTP ࡡ౩⤝ 㜴ᐐࡈࡿࡾࡒࡴ々⿿ࣆ࢚࣭ࢠࡡೳでㄇᑙࡈࡿࡾࠊࡱࡒࠉMMS ࡢ DNA ࡚ࣜ࢞ࣜ࠵ ࡽࠉDNA ୌᮇ㙈ว᩷ࡷᮇ㙈ว᩷࠽ࡻࡦ⬲ሲᇱࢅㄇⓆࡊࠉ々⿿ࣆ࢚࣭ࢠࡡೳでࢅᘤࡀ㉫ࡆ ࡌࠊDNA ഭᐐវུᛮࡷῺᗐវུᛮム㥺ࡡ⤎ᯕࠉ1-113 aa ᩷∞Ⓠ⌟ᰬࡡវུᛮࡢ㔕⏍ᰬ࡞ セ࡙࡛ࢆᕣ↋࠾ࡖࡒࠊࡊࡒࡖ࡙ࠉࡆࡡ᩷∞ࡡ㐛㔖Ꮛᅹ࡞ࡢDNA 々⿿࡞࠽࠷࡙ ࡵಞᚗ࡞࠽࠷࡙ࡵධ㛏Pol31 ࡛࡛ࢆ⇆ࡋᶭ⬗ࢅ⥌ᣚ࡚ࡀࡾࡆ࡛♟ြࡈࡿࡒࠊ࠽ࠉᅒ ୯ G170D ࡛⾪エࡈࡿ࡙࠷ࡾᰬࡢ㧏Ὼࡷ DNA ഭᐐ࡞ᑊࡊ࡙㧏វུᛮ࡚࠵ࡾࡆ࡛▩ࡼࡿ ࡙࠷ࡾⅤን␏ᰬࡡhys2-1 ࡚࠵ࡽ (Sugimoto et al., 1995)ࠉᅂࡡゆᯊ࡚ࡢ㧏វུᛮࡡࢤࣤࢹ ࣭࡛ࣞࣜࡊ࡙⏕࠷ࡒࠊ
Fig. 13 Characterization of mutants overexpressing the 1-113 aa fragment of Pol31. A, Cell cycle analysis by flow cytometry. Cells containing only full length POL31 or 1-113 pol31 on the pKT10L plasmid were grown in medium containing raffinose and galactose until log phase. The cells were then washed and divided into either medium containing raffinose and galactose or into medium containing raffinose and glucose. Samples were taken at the indicated times and analyzed by flow cytometry. B, Histograms of cell cycle distributions as judged by cell morphology. Cell morphology was examined using the cells cultured in the indicated medium for 15 hours. No bud, small bud, and large bud, indicate G1, S, and G2/M phase, respectively. C, Sensitivities of cells expressing the 1-113 aa derivative to temperature and DNA damaging agents. Ten-fold serial dilutions of log phase cells (105, 104, 103, 102 cells) expressing full length Pol31, full length Pol31 having the G170D mutation, or the 1-113 aa derivative
of Pol31 were spotted on SC-Leu plates containing raffinose and galactose. The plates were incubated at 25|C (panel a, c, and d) or at 37|C (panel b). In addition, the plate for panel (c) contained 0.01% MMS and the plate for panel d contained 100 mM HU. The plates were photographed after 4 days incubation.
➠ 4 ⟿ 1-113 aa ᩷ ∞ Ⓠ ⌟ ᰬ ࡡ ⾪ ⌟ ᆵ ࡆࡿࡱ࡚ࡡゆᯊ࠾ࡼN ᮆ❻ 113 aa ࢅ㐛Ⓠ⌟ࡌࡾࡆ࡛࡚ Pol31 ࡡᶭ⬗ࡡ࡛ࢆࢅ┞ ࡚ࡀࡾࡆ࡛♟ြࡈࡿࡒࠊḗ࡞ࡆࡿࢣࢿ࣑୕ࡡࣈ࣓࣭ࣞࢰ࣭࣬ࢰ࣭࣐ࢾ࣭ࢰ࣭㒼า࡞ࡻ ࡖ࡙Ⓠ⌟ࡌࡾpol31 ࢅ⤄ࡲ㎰ࢆࡓࣈࣚࢪ࣐ࢺ්࡚⌟࡚ࡀࡾ࠾ࢅ᳠ゞࡊࡒࠊ ࡐࡡ⤎ᯕࠉ⏍⌦Ⓩ㔖ࡡPol31 1-113 aa ᩷∞ࢅⓆ⌟ࡌࡾ⣵⬂ࡢ 5-FOA ྱ᭯ᇰᆀ୕࡚ࡵ㐛 Ⓠ⌟ࡡሔྙ࡛⇆ᵕ࡞ࢤࣞࢼ࣭ࢅᙟᠺࡊࡒࠊୌ᪁ࠉ1-96 aa ᩷∞Ⓠ⌟⣵⬂ࡢࠉ㐛Ⓠ⌟ࡡ࡛ ⇆ᵕ࡞ࢤࣞࢼ࣭ࢅᙟᠺࡊ࠾ࡖࡒࠊࡊࡒࡖ࡙ࠉ⣵⬂⑵る⬗ࡡ᭯↋࡞ࡗ࠷࡙ࡢࠉ㐛Ⓠ⌟ ࡈࡎࡒሔྙ࡛⇆ᵕࡡ⤎ᯕᚋࡼࡿࡒ (Fig. 14A)ࠊ ḗ࡞ⷾវུᛮ࡛Ὼᗐវུᛮム㥺ࢅ⾔ࡖࡒࠊࡌࡾ࡛ࠉ1-113 aa ᩷∞Ⓠ⌟ᰬࡢⷾ࡞ᑊࡌࡾ វུᛮࡢ㔕⏍ᰬ࡛࡛ࢆንࢂࡼ࠷ࡡ࡞ᑊࡊ࡙ࠉ㧏Ὼ࡞ᑊࡌࡾវུᛮࢅ♟ࡌࡆ࡛ࢂ࠾ ࡖࡒ(Fig. 14B)ࠊ ḗ࡞㧏Ὼវུᛮࡡリ⣵࡞ࡗ࠷࡙ㄢࡾࡒࡴ࡞⣵⬂࿔ࡡᣪິࢅびᐳࡊࡒࠊ᪁Ἢ࡛ࡊ࡙ࡢ Pol31 ධ㛏ࠉG170D 偆hys2-1 假ࠉ1-113 aa ᩷∞Ⓠ⌟ᰬࢅࠍ25 0C ࡚ᑊᩐ⑵るࡱ࡚ᇰ㣬ᚃࠉ 25 0C ࠾ࡼ 37 0C ࡞Ὼᗐࢅ⛛ࡊࠉࡐࡡᚃ 3 㛣࠾ࡼ 8 㛣ࡱ࡚ 1 㛣ࡇ࡛࡞⣵⬂ࢅࢦࣤࣈࣛ ࣤࢡࡊࠉࣆ࣭ࣞࢦࢹ࣒ࢹ࣭ࣛ࡞ࡻࡽゆᯊࡊࡒࠊࡐࡡ⤎ᯕࠉ㧏Ὼ᮪௲࡚㛣⤊ࡾ࡞ࡗࡿ 1-113 aa ᩷∞Ⓠ⌟⣵⬂࡚ࡢ S ࠾ࡼ G2/M ࡡ✒ࡡലྡྷᙁࡂࡖ࡙࠷ࡂᵕᏄびᐳࡈ ࡿࡒ (Fig. 14C panel b)ࠊࡆࡡ✒ࡢࠉhys2-1 ᰬࡡ S ᚃ࠾ࡼ G2/M ࡡ✒࡞セࡾ࡛ 1-113 aa ᩷∞Ⓠ⌟ᰬ࡚ࡢࡻࡽ S ࡡ✒ࡡലྡྷᙁ࠾ࡖࡒࠊ ࡈࡼ࡞S ࡡ㐅⾔ࡡ㏷ᗐࡡንࢅビ౮ࡌࡾࡒࡴ࡞ᑊᩐ⑵るࡱ࡚ᇰ㣬ࡊࡒ⣵⬂ࢅ α-factor ࡚G1 ࡞⇆ㄢࡊࠉࡐࡡ⇆ㄢࢅゆ㝎ࡊ࡙ᇰ㣬ࢅ්㛜ࡊࠉS ࡡ㐅⾔ࢅࣆ࣭ࣞࢦࢹ࣒ࢹ࣭ࣛ ࡚࣓ࢼࢰ࣭ࡊࡒࠊࡐࡡ⤎ᯕࠉ≁࡞⇆ㄢࢅゆ㝎ࡊࡒ30 ฦᚃࡡ G2/M ࡡ⣵⬂ᩐ࡞Ἰ┘ࡌࡾ࡛ࠉ 1-113 aa ᩷∞Ⓠ⌟ᰬ࡚ࡢ㔕⏍ᰬ࡛ hys2-1 ᰬࡡ୯㛣࡚࠵ࡖࡒࠊࡌࢂࡔ 1-113 aa ࡚ S ࡡ㐅⾔ 㐔ࡂࡖ࡙࠷ࡒ (Fig 15D)ࠊ 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ㧏Ὼ࡞ᑊࡌࡾវུᛮ࡛ S ࡡ㐔ᘇࢅెࡎ࡙⩻࠻ࡾ࡛ࠉ1-113 aa ᩷∞ Ⓠ⌟ᰬࡢ㗢ᆵ㙈୕࡞ഭࢅㄇᑙࡊ࠷ሔྙ࡚ࡵⱕᖱ々⿿ࡡ㐅⾔ࡡᏭᏽᛮ࡞Ḗࡄ࡙࠷ࡾ࡛⩻
࠻ࡼࡿࡒࠊࡆࡡⅴᅄࡢࠉ⇍ຊࢂࡾࡆ࡛࡚々⿿ࣆ࢚࣭ࢠ㢎⦶࡞ቪࡿࠉPol δ ࡐࡡࡵࡡࡡᏭ ᏽᛮࢂࡿࡾࡒࡴ࡚࠵ࡽࠉࡐࡡ⤎ᯕࠉS ࡡ㐔ᘇ㉫ࡆࡾࡡ࡚࠷࠾࡛᥆ῼࡈࡿࡒࠊ
Fig. 14 Characterization of cells expressing the 1-113 fragment of Pol31 from its own promoter (1-113 aa pol31 cell) on a single copy plasmid. A, Effect of deletion of full length POL31. Cells carrying the plasmid indicated in panel (a) and the pRS316-P31 (URA3) plasmid were streaked on SC-Ura-Leu plates (panel b) or SC-Leu plates containing 5-FOA (panel c), incubated at 25|C, and then photographed after 5 days. B, Sensitivities of the pol31 cells expressing the 1-113 aa derivative to temperature and DNA damaging agents. Ten-fold serial dilutions of log phase cells (105, 104, 103, 102 cells) expressing full length Pol31, full length Pol31 having the G170D mutation, or the 1-113 aa derivative of Pol31 were spotted on SC-Leu plates containing raffinose and galactose. The plates were incubated at 25|C (panel a, c, and d) or at 37|C (panel b). In addition, the plate for panel (c) contained 0.01% MMS and the plate for panel d contained 100 mM HU. The plates were photographed after 4 days incubation. C, Cell cycle analysis by flow cytometry. Cells containing only full length POL31 or G170D pol31 or 1-113 aa pol31 on the pRS315 plasmid were grown in medium until log phase at 25|C. The culture temperature was then shifted to 37|C. Samples were taken at the indicated times and analyzed by flow cytometry. D, S phase progression of 1-113 aa pol31 cells. The cells were synchronized by a-factor at 25|C, released from G1 phase. Samples were taken at the indicated times.
pol31 ࡡን␏ࡢࠉ々⿿ࣆ࢚࣭ࢠࡡೳで㉫ࡆࡖࡒ㝷࡞්㛜ࡡࡒࡴ࡞഼ࡂࢰࣤࣂࢠ㈹ࢅࢤ࣭ ࢺࡌࡾ㐿ఎᏄ࡛㐿ఎᏕⓏ┞பష⏕࠵ࡾࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Branzei et al., 2002; Vijeh Motlagh et al., 2006)ࠊࡐࡆ࡚ࠉ1-113 aa ᩷∞Ⓠ⌟࡛࠷࠹ን␏࡛ࡐࡿࡼࡡ㐿ఎᏄ◒ቪ࡛ࡡ㔔 ን␏ࢅࡵࡗᰬࢅష⿿ࡊࠉࡐࡡ⑵る⬗ࢅㄢࡒࠊ
࠽ࠉRad18 ࡢ๑㏑ࡊࡒ Post Replication Repair ࡞࠽࠷࡙ PCNA ࡡ࣓ࢿࣗࣄ࢞ࢲࣤ࡞഼ ࡂE3 ࡚࠵ࡾ (Fig. 15)ࠊࡱࡒࠉMms2 ࡢ PRR ࡡ࠹ࡔࠉ࣎ࣛࣗࣄ࢞ࢲࣤ࡞㛭ࢂࡾ E2 ࡡᵋᠺ ฦᏄࡡୌࡗ࡚࠵ࡾ (Fig. 15)ࠊୌ᪁ࠉMgs1 ࡢ๑㏑ࡊࡒ WRNIP1 ࡡฝⰾ㓕ザ࣓࣌ࣞࢡ࡚࠵ࡾࠉ ࡐࡡ㐿ఎᏄ◒ቪRAD6 ◒ቪ࡛ࡡ㔔◒ቪᰬ࡚ྙᠺ⮬ほ࡛ࡾࡆ࡛ (Hishida et al., 2002) ࡷ hys2-1 ን␏࡛ࡡ㐿ఎᏕⓏ┞பష⏕࠾ࡼ Mgs1 ࡡᙲ࡞ࡗ࠷࡙々⿿ࣆ࢚࣭ࢠೳでࡊࡒ㝷ࠉ Rad18 ࡞౪Ꮛࡊࡒ⤊㊨࡛ࡢื⤊㊨࡚々⿿ࣆ࢚࣭ࢠࡡ㐅⾔ࡡ්㛜࡞഼ࡂࡡ࡚ࡢ࠷࠾࡛᥆ῼ
ࡊ࡙࠷ࡾ (Branzei et al., 2002)ࠊ
௧୕ࡡࡻ࠹ሒ࿈࠾ࡼ Pol31 ࡡ C ᮆ❻ഁࡡḖ࡛ࡆࡿࡼࡡ㐿ఎᏄࡡ◒ቪ࡞ࡻࡖ࡙々⿿ࣆ ࢚࣭ࢠࡡᏭᏽᛮࡈࡼ࡞ࢂࡿࠉ⣵⬂⑵る⬗࡞ᙫ㡢ࢅ࠻ࡾࡆ࡛ࡈࡿࡒࠉࡆࡿ ࡼ㐿ఎᏄࡡ◒ቪ࡞ࡻࡖ࡙1-113 aa ᩷∞Ⓠ⌟ᰬࡡ⣵⬂⑵る⬗ࡢࢂࡿࡾࡆ࡛ࡢ࠾ࡖࡒ (Fig. 16, panel f)ࠊ
࡞ࡵ࣎ࣛࣗࣄ࢞ࢲࣤࡡE3 ࢅࢤ࣭ࢺࡌࡾ RAD5ࠉഭࡽ㉲࠻ᆵ DNA 々⿿㓕⣪ Pol ζ ࡡよ፳ࢦࣇࣗࢼࢴࢹࢅࢤ࣭ࢺࡌࡾREV3ࠉ⤄ࡲᥦ࠻ཬᚺࡡᢒโ࡞㛭ࢂࡾࣉ࣭ࣛ࢜ࢭࢅࢤ࣭ࢺ
ࡌࡾ SRS2 偆hys2-1 ࡛ࡡ㔔ን␏࡚⑵る⬗΅ᘽࡌࡾ假 々⿿ࣆ࢚࣭ࢠࡡ㐅⾔ࡡ⥌ᣚ࡞
㛭ࢂࡾ࡛ࡈࡿࡾ々ᩐࡡ㐿ఎᏄ⛸࡛ࡡ㔔ን␏ᰬࢅష⿿ࡊࡒࠉ⣵⬂⑵る⬗ࢂࡿࡾ ࡆ࡛ࡢ࠾ࡖࡒ (data not shown)ࠊ
ୌ᪁ࠉPol δ 々ྙమࡡࢦࣇࣗࢼࢴࢹ࡚࠵ࡾ Pol32 ࢅḖࡊࡒࠉ1-113 aa ᩷∞Ⓠ⌟ᰬࡡ⑵る ⬗ࡢࢂࡿࡒ (Fig. 16)ࠊࡊࡒࡖ࡙ 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ⑵る࡞ࡢ Pol32 ࢦࣇࣗࢼࢴࢹᚪ こ࡚࠵ࡾࡆ࡛ࢂ࠾ࡖࡒ (Fig. 16, panel f)ࠊ
Fig. 15 Post replication repair In Saccharomyces cerevisiae, genes belonging to theRAD6 epistasis group are
responsible for the PRR pathway. RAD6, RAD18, RAD30, RAD5, REV3, and REV7 are majormembers of this group. DNA gaps caused by replication stallingare filled by translesion synthesis either in an error-freeor an error-prone mode depending on the context of the DNA damage.
Fig. 16 Effect of deletion of various genes in pol31 cells expressing the 1-113 aa derivative of Pol31. The cells disrupted in the indicated gene (panel a and b) carried either the single copy plasmid encoding full length Pol31 (panel c and d) or the 1-113 derivative of Pol31 (panel d and f) and the pRS316-P31 (URA3) plasmid were streaked on SC-Ura-Leu plates (panel c and d) or SC-Leu plates containing 5-FOA (panel e and f), incubated at 25|C, and then photographed after 5 days.
➠ 5 ⟿ PCNA / Mgs1 㐛 Ⓠ ⌟ ࡡ ᙫ 㡢
ࡆࡿࡱ࡚ࡡPOL31 ࡡⅤን␏ᰬࡡゆᯊ࡞ࡻࡽࠉMgs1 ࡡ㐛Ⓠ⌟࡞ࡻࡖ࡙⣵⬂ࡡᠺ⫩ᝇࡂ ࡽࠉMMS វུᛮ⑵ຊࡌࡾࡆ࡛ࠉPCNA ࡡ㐛Ⓠ⌟࡞ࡻࡖ࡙ HU ࡷ MMS វུᛮ⑵ຊ ࡌࡾ࡛࠷࠹ሒ࿈࠵ࡾ (Branzei et al., 2002; Vijeh Motlagh et al., 2006)ࠊࡐࡆ࡚ࠉ1-113 aa ᩷∞ Ⓠ⌟ᰬ࡞ࡗ࠷࡙ Mgs1ࠉPCNA ࢅ㐛Ⓠ⌟ࡈࡎࠉࡐࡡᙫ㡢ࢅㄢࡒࠊ࠽ࠉMGS1 ࡢ GAL1 ࣈ࣓࣭ࣞࢰ࣭โᚒࡡࣈࣚࢪ࣐ࢺ୕࡞ࢤ࣭ࢺࡈࡿ࡙࠷ࡾࡒࡴࠉgalactose ྱ᭯ᇰᆀ࡚ࡡࡲ㐛Ⓠ ⌟ࡌࡾࠊ
ࡐࡡ⤎ᯕࠉMgs1 ࡡ㐛Ⓠ⌟ࡢ 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ DNA ഭᐐ㟸῟ຊࠉ࠽ࡻࡦ HU࣬ MMS ῟ຊࡡ⑵る⬗ࢅୖࡈࡎࡒ (Fig. 17A, panel b)ࠊୌ᪁ࠉPCNA ࡡ㐛Ⓠ⌟ࡢࡆࡡᰬࡡ HU࣬MMS ῟ຊࡡ⣵⬂⑵る࡞ࡡࡲᝇᙫ㡢ࢅ࠻ࡒ (Fig. 17B)ࠊ
Fig. 17 Overexpression of Mgs1 or PCNA causes further defects in 1-113 aa pol31 cells. A, Ten-fold serial dilutions of log phase cells (105, 104, 103, 102 cells) expressing full length Pol31 or the 1-113 aa derivative of Pol31 were spotted on SC-Leu plates containing glucose (upper panels) or galactose (lower panels) with the indicated concentration of HU or MMS. MGS1 was carried on a plasmid containing the GAL1 promoter. Cells were incubated at 25|C and then photographed after 4 days. B, Ten-fold serial dilutions of log phase cells (105, 104, 103,
102 cells) expressing full length Pol31 or the 1-113 aa fragment of Pol31 were spotted on SC-Leu plates containing the indicated concentration of HU or MMS. POL30 (PCNA) was overexpressed from its own promoter on a multicopy plasmid. Cells were incubated at 25|C and photographed after 4 days.
➠ 6 ⟿ N ᮆ ❻ ഁ ┞ ப ష ⏕ 㡷 ᇡ ࡡ ≁ ᏽ ࡆࡿࡱ࡚ࡡゆᯊ࠾ࡼ i) 1-113 aa 㡷ᇡࡡⓆ⌟ࡡࡲ࡚⑵るྊ⬗࡚࠵ࡾࡆ࡛ (➠ 2 ⟿) ii) 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ HU࣬MMS វུᛮࡢ㔕⏍ᰬ࡛࡛ࢆንࢂࡼ࠷ࡆ࡛ (➠ 3 ⟿ࠉ➠ 4 ⟿) iii) 1-113 aa ᩷∞Ⓠ⌟ᰬࡡ⏍Ꮛࡢ Pol32 ࡞౪Ꮛࡊ࡙࠷ࡾࡆ࡛ (➠ 4 ⟿) ᪺ࡼ࠾࡞ࡖࡒࠊ௧୕ࡡ⤎ᯕ࡛ 3 ࡗࡡࢦࣇࣗࢼࢴࢹࡐࡿࡑࡿࡡ㐿ఎᏄḖᰬࡡ⾪⌟ᆵࢅ ⩻៎ࡌࡾ࡛ࠉPol31 ࡢ 1-113 aa ࡡ㡷ᇡ࡚ Pol3ࠉPol32 ࡡཫ᪁࡞⤎ྙࡊࠉ1-113 aa ࡢࣉࢷࣞ 3 㔖మᙟᠺ࡚ࡀࡾࡡ࡚ࡢ࠷࠾࡛⩻࠻ࡼࡿࡒࠊࡐࡆ࡚ࠉࡆࡡ௫ㄕࢅඞỷ㜾Ἢࢅ⏕࠷࡙᳠ チࡊࡒ (➠ 1 ⟿)ࠊ ࡐࡡ⤎ᯕࠉPol3ࠉPol32 ࡛ࡵ࡞ 1-113 aa ᩷∞࡛භỷ㜾ࡌࡾࡆ࡛ࢂ࠾ࡖࡒࠊୌ᪁ࠉ⣵⬂ࡡ ⑵るࢅᨥᣚ࡚ࡀ࠷1-96 aa ᩷∞ࡢ Pol3ࠉPol32 ࡛ࡵ࡞භỷ㜾ࡊ࠾ࡖࡒ (Fig. 18A)ࠊࡱࡒࠉ C ᮆ❻ഁࡢ Pol3 ࡛ࡡࡲභỷ㜾ࡊࠉPol32 ࡛ࡢභỷ㜾ࡊ࠾ࡖࡒࠊࡊࡒࡖ࡙ࠉN ᮆ❻ 1-113 aa ᩷∞ࡢPol δ 々ྙమࡡᙟᠺࡡࡒࡴ࡞ᚪこ࠾ࡗ༎ฦ㡷ᇡ࡚࠵ࡾࡆ࡛ࢂ࠾ࡖࡒࠊ
Fig. 18 Physical interaction between the 1-113 fragment of Pol31 and both Pol3 and Pol32. A, Immunoprecipitaton of Polδ subunits. Extracts were prepared from Pol32-FLAG and Pol3-FLAG cells transformed with pKT10-P31, pKT10L-p31-N10 (1-113 a.a.), pKT10-p31-N11 (1-96 a.a.), or pKT10L (empty). HA-tagged proteins were immunoprecipitated with anti-HA antibody-conjugated agarose beads as described under Experimental Procedures. Immunoprecipitants were fractionated by SDS-PAGE and analyzed by immunoblotting with an anti-FLAG monoclonal antibody and anti-HA antibody. I.B., immunoblotting; I.P., immunoprecipitation. (a) Pol3 (b) Pol32 B, Summary of coimmunoprecipitation analysis.
➠
4 ❮ ⩻ᐳ
Pol31 N ᮆ❻ഁࡡᶭ⬗ᮇ◂✪࠾ࡼࠉධ㛏 487 aa ࡡ Pol31 ࡡ࠹ࡔ N ᮆ❻ 1-113 aa ࡡࡲ࡚⣵⬂⑵るࢅ⥌ᣚࡊࠉDNA 々⿿࣬ಞᚗࢅ⾔࠹ࡆ࡛࡚ࡀࡾPol δ 々ྙమᙟᠺࡈࡿࡾࡆ࡛ࡢࡋࡴ࡙᪺ࡼ࠾࡞ࡖࡒࠊ
Pol31 ࡢධ㛏࡞ࢂࡒࡽ I-X ࡡ 10 ࡡ࣐ࢿ㓗㒼าಕᏋ㡷ᇡ偆ࢺ࣒ࣤ假ࢅ᭯ࡌࡾࠊ1-113 aa ࡢࠉࡆࡡ࠹ࡔ I ࡛ II ࡡ༖ฦࡊ࠾ྱࡱࡿ࡙࠷࠷ (Fig. 19 panel a)ࠊᮇ◂✪࡞࠽࠷࡙ I ࡛ II ࡡࢺ࣒ࣤ Pol3 ࠽ࡻࡦ Pol32 ࡡ୦᪁ࡡࢦࣇࣗࢼࢴࢹ࡛⤎ྙࡌࡾࡆ࡛♟ြࡈࡿࡒࠊI ࡛ II ࡡࢺ࣒ࣤࢅ㐛Ⓠ⌟࣊ࢠࢰѸࢅ⏕࠷ࡍ࡞⏍⌦Ⓩ㔖ࢅⓆ⌟ࡈࡎࡒ㝷偆Single copy vector ࢅ⏕࠷ࡒሔྙ假࡞ࡢࠉ⣵⬂ࡢῺᗐវུᛮࢅ♟ࡊG2/M ࡞✒ࡊࡒࠊᚉࡖ࡙ࠉI ࡛ II ࡡࢺ࣒ ࣤࡡࡲ࡚ࡢPol δ 々ྙమ᮪௲࡞ࡻࡖ࡙ࡢᏭᏽ≟ឺ࡞㝏ࡾ࡛⩻࠻ࡼࡿࡾࠊࡆࡆ࡚ XI-X ࡡࢺ࣒ࣤ Pol3 ࡛⤎ྙ࡚ࡀࡾࡆ࡛ࢅ⩻៎ࡌࡾ࡛ࠉXI-X ࢺ࣒ࣤࡢ⣵⬂ࡡ⏍Ꮛ࡞ᚪ㡪࡚ ࠷ࡵࡡࡡࠉࡻࡽᏭᏽPol δ 々ྙమࡡᙟᠺ࡞ᚪこ࡚࠵ࡾ࡛⩻࠻ࡼࡿࡾ (Fig. 19 panel b; Fig. 20)ࠊ
᭩㎾ࣃࢹPol δ ࡡ⏍ᏕⓏゆᯊ࠾ࡼࠉPol δ ࡢ々⿿୯࡞ RFCࠉPCNA ࡛ゆ㞫࣬㞗ྙࢅ⧖ࡽ㏁ ࡊࡗࡗ DNA ྙᠺࢅ⾔ࡖ࡙࠷ࡾ࡛࠷࠹࣓ࢸࣜᥞ♟ࡈࡿࡒ 偆Masuda et al., 2007假ࠊࡆࡡ࣓ࢸ ࣜ࡞ᚉ࠻ࡣࠉ㧏Ὼ࡛࠷࠹ࢪࢹࣝࢪ᮪௲ୖ࡚ࡢPol δ ࡢ々⿿ࣆ࢚࣭ࢠ࠾ࡼ⬲ⴘࡊࠉࡐࡡᚃ࡞々 ⿿ࣆ࢚࣭ࢠ࡞්⤎ྙࡌࡾ࡛⩻࠻ࡼࡿࡾࠊࡆࡡࡆ࡛ࢅ⩻៎ࡌࡾ࡛ࠉ1-113 aa ᩷∞ 偆I ࡛ II ࡡ ࢺ࣒ࣤࡡࡲ假 ࢅⓆ⌟ࡌࡾ⣵⬂࡚ࡢS 㐅⾔୯ࠉ㧏Ὼ᮪௲ୖ࡚Ꮽᏽ Pol δ 々ྙమ㢎 ⦶࡞々⿿ࣆ࢚࣭ࢠ࠾ࡼ⬲ⴘࡊࠉࡈࡼ࡞᩺ࡒPol δ 々ྙమࡡ්⤎ྙ࡞ࡵᨥ㝸⏍ࡋࠉࡐࡡ⤎ ᯕ࡛ࡊ࡙Ὼᗐវུᛮࢅ♟ࡌྊ⬗ᛮ⩻࠻ࡼࡿࡾࠊ
Fig.19 A model of Pold complex in this study (a) Sequence alignment of the 1-113 aa regions of Pol31 among various species. (b) A model of the interactions of Pol31 with Pol3 and Pol32.
Fig. 20 Summary of subunit interaction Domains for subunit interaction are summarized (Garcia et al ., 2004; Johansson et al., 2004).
Pol31 C ᮆ❻ഁࡡᶭ⬗
Pol δ ࡡຐᅄᏄ࡚࠵ࡾ PCNA ࡢム㥺⟮හࡡ Pol δ ࡡఘ㛏ཬᚺࢅⓏ࡞ಀ㐅ࡊࠉ⣵⬂ࡡ⏍ ⫩ࠉDNA 々⿿࡞ᚪ㡪࡚࠵ࡾࠊฝⰾ㓕ザࡡ Pol δ 々ྙమࡡࢦࣇࣗࢼࢴࢹࡡ࠹ࡔ PCNA ࡛ࡡ ⤎ྙ᪺☔࡞♟ࡈࡿ࡙࠷ࡾࡡࡢ⏍Ꮛ࡞ᚪ㡪࡚ࡢ࠷ Pol32 ࢦࣇࣗࢼࢴࢹࡡࡲ࡚࠵ࡾࠊࡊ࠾
ࡊࠉム㥺⟮හ࡚Pol3/Pol31 ࡡ 2 ࡗࡡࢦࣇࣗࢼࢴࢹࡡࡲ࠾ࡼᵋᠺࡈࡿࡾ Pol δ 々ྙమ 㐛 ࡞Ꮛᅹࡌࡾ࡛ࠉPCNA ࡞ࡻࡽ DNA ྙᠺಀ㐅ࡈࡿࡾࡻ࠹࡞ࡾࠊࡊࡒࡖ࡙ࠉPol32 㟸Ꮛ ᅹ࡞ࡢPol3 ࠵ࡾ࠷ࡢ Pol31 ࡡࡔࡼ࠾ࡡࢦࣇࣗࢼࢴࢹ PCNA ࡛┞பష⏕ࡌࡾ࡛ࡈ ࡿ࡙࠷ࡾࠊᮇ◂✪࡚ࡢPol31 ࡡ C ᮆ❻Ḗ (114-487 aa 㡷ᇡࡡḖ) ࡛ Pol32 ࡡ⇆Ḗ ⣵⬂࡞ほࢅࡵࡒࡼࡌࡆ࡛ࢅ♟ࡊࡒࠊୌ᪁ࠉ௧๑ᙔ◂✪ᐄ࡚⾔ࢂࡿࡒPol31 ࡡಕᏋࡈࡿࡒ࣐ ࢿ㓗よᇱࡡណ⩇ࢅゆᯊࡌࡾ◂✪࡞ࡻࡽࠉC ᮆ❻ഁࡡ࣐ࢪࢬࣤࢪን␏ (R256AࠉGN261AAࠉ D414AࠉD416A) ࡛ POL32 㐿ఎᏄ◒ቪࡡ㔔ን␏࡞ࡻࡖ࡙⣵⬂⑵る⬗ⴥࡊࡂୖࡌࡾࡆ ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Vijeh Motlagh et al., 2006) (Fig. 21 panel g)ࠊᮇ◂✪ࡡ⤎ᯕ࡛ྙࢂࡎ࡙⩻ ࠻ࡾ࡛ࠉPol31 ࡡ 114-487 aa ࡡ㡷ᇡ࡞ࡢ Pol32 㟸Ꮛᅹ࡞ Pol d々ྙమ࡛ PCNA ࡛ࡡ┞பష ⏕ࢅ⥌ᣚࡌࡾᙲ࠵ࡾྊ⬗ᛮ⩻࠻ࡼࡿࡒࠊࡌࢂࡔࠉPol31 ࡡ C ᮆ❻ഁࡡḖࡱࡒࡢ Ⅴን␏࡞ࡻࡖ࡙Pol d ࡛ PCNA ࡛ࡡ┞பష⏕◒⥚ࡊࠉDNA ྙᠺ࡞ᨥ㝸ࢅࡀࡒࡊࡒ࡛᥆ῼ ࡈࡿࡒࠊࡆࡡ᥆ῼࢅ᳠チࡌࡾࡒࡴ࡞Pol31 ࡡ 1-113aa Pol32 ࡡ PIP box ን␏偆Pol32 ࡡ PCNA ┞பష⏕㡷ᇡ࡚࠵ࡾC ᮆ❻ PIP box Ḗ假࡛භᏋࡊ࠹ࡾ࠾ྫྷ࠾ㄢࡾࡆ࡛ࡢᚃࡡㄚ㢗࡚࠵ ࡾࠊ
Fig. 21 Schematic models of Pol d comprising various Pol31 mutant derivatives. Left panels (panel a, b, c, and d) show the putative subunit constitution of Pold comprising various Pol31 mutants in the presence of both intact Pol3 and Pol32. Right panels (panel e, f, and g) show the putative subunit constitution of Pold comprising various Pol31 mutants in the presence of intact Pol3 but in the absence of Pol32. Points mutation presented on panel c and g were described in our previous study.
ᚃࡡᒈ᭻
ᗆㄵ࡚㏑ࡒࡻ࠹࡞ Pol31 ࡡᶭ⬗ࢅ᥀ࡾ◂✪ࡢࡆࡿࡱ࡚࡛ࢆࡈࡿ࡙࠷࠾ࡖࡒࠊ ࡊ࠾ࡊࠉ᭩㎾࡞ࡖ࡙ Pol31 ࡞㛭ࡌࡾሒ࿈ࡵᩋずࡈࡿࡾࡻ࠹࡞ࡽࡗࡗ࠵ࡾࠊᐁ㝷ࠉPol31 Yra1 ࡛ฝⰾ㓕ザ⣵⬂හ࡚┞பష⏕ࡊ࡙࠷ࡾ࡛࠷࠹ሒ࿈ࡈࡿࡒ (Swaminathan et al., 2007)ࠊ⮾࠷ࡆ࡛࡞ࠉ
(i) yra1 ◒ቪᰬࡢ㧏Ὼ᮪௲࡚ G1 ⇆ㄢᚃ S ࡞㐅⾔࡚ࡀࡂࡾࠊ (ii) Yra1 ࡢ replication origin ࡞⤎ྙࡌࡾࠊࡱࡒ Yra1 ࡢ Dia2 ࡛⤎ྙࡌࡾࠊ (iii) dia2 㐿ఎᏄ◒ቪ࡛ YRA1 ࡡ C ᮆ❻Ḗ㐿ఎᏄ࡛ࡡ㔔ን␏ᰬࡢ⮬ほ࡚࠵ࡾࠊ 偆Dia2 ࡢ Skp1/Cdc53/F-box ubiquitin ligase 々ྙమ>SCF 々ྙమ@ࡡᵋᠺᅄᏄࡡୌࡗ ࡚࠵ࡾࠊSCF 々ྙమࡢࣗࣄ࢞ࢲࣤ࡞ࡻࡾࢰࣤࣂࢠ㈹ฦゆ࡞ࡻࡖ࡙⣵⬂࿔ࢅ ㄢ⟿ࡌࡾࡆ࡛▩ࡼࡿ࡙࠷ࡾ假
௧๑࡞ dia2 ༟≺◒ቪᰬ HUࠉMMSࠉCamptothecin ࡡ DNA ഭᐐ࡞ᑊࡊ࡙វུᛮࢅ ♟ࡌ࡛࡛ࡵ࡞ࠉDNA 々⿿࡞Ḗࢅ♟ࡌࡆ࡛ሒ࿈ࡈࡿ࡙࠷ࡾ (Blanke et al., 2006)ࠊࡆࡿࡼ ࡡሒ࿈ࢅࡨࡱ࠻ࡾ࡛ࠉᮇ◂✪࡚ࡢ࠵ࡱࡽゆᯊ࡚ࡀ࠾ࡖࡒ Pol31 ࡡ C ᮆ㡷ᇡ偆III-X ࢺ࣒ ࣤ假ࡡฝⰾ㓕ザ⣵⬂හࡡᶭ⬗ࠉࡆࡆ࡚㏑ࡒ Yra1 ࡷ Dia2 ࡛ࣛࣤࢠࡊ࡙࠷ࡂࡡ࡚ࡢ ࠷࠾࡛⩻࠻ࡼࡿࡾࠊ ㎾ᖳࠉ々⿿ࡡೳで࡛ࡐࡡ්㛜ࡡ㝷࡞ࢣࢿ࣑ࡡ්⥽ࢅఔ࠹ኬࡀን␏⏍ࡋࠉࡐࡿ㐿ఎ ࡡⅴᅄ࡛ࡾሔྙኣᩐᏋᅹࡌࡾྊ⬗ᛮሒ࿈ࡈࡿࡒ偆ᗆㄵ假ࠊࡆࡡ࠾࡚ࠉᙔ◂✪ᐄࡡ ௧๑ࡡ◂✪偆ฝⰾ㓕ザࡡ㐿ఎᏕⓏゆᯊ假࡞ᇱࡘࡀࠉPol δ ࡐࡡ୯ᚨⓏᙲࢅᢰ࠹ྊ⬗ᛮ ᣞᦤࡈࡿ࡙࠷ࡾࠊࡈࡼ࡞ᙔ◂✪ᐄࡡ◂✪࡞ࡻࡽࠉࣃࢹ WRNIP1 偆ࣃࢹ᪡⩹ⅴᅄ㐿ఎᏄ ⏐∸ WRN ࡛⤎ྙࡌࡾࢰࣤࣂࢠ㈹假ࣃࢹ Pol δ ࡡὩᛮࢅム㥺⟮හ࡚ಀ㐅ࡌࡾࡆ࡛♟ࡈࡿ
