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Ȁƺ
ɝƙ ................................................................................................................................. 1Ǵɱɢ .......................................................................................................................... 4Ȝ 1 Ț ōɶ ..................................................................................................................... 5Ȝ 2 Ț REiʢçƈ¿ːREiĚėĺ+ĭİçJkP +ɨȠ ................... 10Ȝ 1 ȡ Ƚƞ.............................................................................................................. 10Ȝ 2 ȡ ƕǈ.............................................................................................................. 12Ȝ 3 ȡ ȱƭ'ȺĹ ................................................................................................... 13Ȝ 4 ȡ ȱɶ.............................................................................................................. 23Ȝ 3 Ț REiʢçƈ¿ːREiĚėĺBü7 DNA ƨʧ+ƽ6+ɨȠ ........... 24Ȝ 1 ȡ Ƚƞ.............................................................................................................. 24Ȝ 2 ȡ ƕǈ.............................................................................................................. 27Ȝ 3 ȡ ȱƭ'ȺĹ ................................................................................................... 32Ȝ 4 ȡ ȱɶ.............................................................................................................. 42Ȝ 4 Ț Oz *ĺ>Ěė+õ=ʎ6ɥƫ ...................................................................... 44Ȝ 1 ȡ Ƚƞ.............................................................................................................. 44Ȝ 2 ȡ ıˋƕǈ ...................................................................................................... 46Ȝ 3 ȡ ȱƭ'ȺĹ ................................................................................................... 56
Ȝ 4 ȡ ȱɶ.............................................................................................................. 76Ȝ 5 Ț Oz Bü7Ěėĺ+ĭİŪ+ DNA ©ʨóŦ*

>Ŗʿ ............................... 78Ȝ 1 ȡ Ƚƞ.............................................................................................................. 78Ȝ 2 ȡ ıˋƕǈ ...................................................................................................... 80Ȝ 3 ȡ ȱƭ'ȺĹ ................................................................................................... 85Ȝ 4 ȡ ȱɶ.............................................................................................................. 96

Ȝ 6 Ț Oz ʔȳʠÖ+ DNA z~]óŦ*ĺ>Ŗʿ ...................................... 97Ȝ 1 ȡ Ƚƞ.............................................................................................................. 97Ȝ 2 ȡ ıˋƕǈ ...................................................................................................... 99Ȝ 3 ȡ ȱƭ'ȺĹ ................................................................................................. 102
Ȝ 4 ȡ ȱɶ............................................................................................................ 112
Ȝ 7 Ț ȴž ................................................................................................................. 114
ñȺƓǩ ...................................................................................................................... 118
ɸʍ ............................................................................................................................. 129

ɝƙ
DNA ,­Íğ<+ƶ)ʢçZfZ*ŋ*ơ?%REiƣ9ʢçBö:!%ǯųREiʢçƈ¿*ĺ>Eei

:REi+ɲõ=ʎ6,ƣȮǼ*REi&> G:C-T:A fZmY=Ǧ* DNA Ěė+<b|:XfX1+ǞȘǠĞǵ

;/G:C-C:G fZmY+ïČ')>

?<+ǞȘǠĞǵ,ƶ)ʝ¨Ĩ&ƳÔ?%=°'%CŶÛʝ¨Ĩ&> p53 ʝ¨Ĩ:Cʝ¨Ĩ&> K-ras ʝ¨Ĩ<?>Ǧ*Cʝ¨Ĩ&> K-ras ʝ¨Ĩ+ǍŪēĞǵ+ïČ')> codon 12 *

> G:C-C:G fZmY: G:C-T:A fZmY+ǻǯ,ġɄC:ɆCȼC'C&ˏ˅Ŏ*ƳÔ?%"øȓ+>+8?<+ǞȘǠĞǵ+ǻǯ~Mi[}*#%ȊȖ>',ǞȘǠĞǵ*;=ǯ>C+ǻǹ~Mi[}+ɥƛ*

%ʣ

ɝ&>
ƶ)Ʃ¦&ǯų>¤ɘǼ)REiʢçƈ¿&> 8 LO^REi
(8-oxoG) ,ƣ9ȊȖʕC& =8-oxoG , G:C-T:A fZmYBőʂƈ¿&>'Ⱥ<?%>ƕ8-oxoG ,REi',ĚėĺBŕų&Ȃɚʧ½*REiƂÈ?)#5=G:C-C:G fZmY,¢+REiʢçƈ¿*;"%őʂ?>'ȺREi+ʢç<?>

;/ 8-oxoG +<)>ʢç*;=ǯų>G|a_ (Iz) ßǄÕɥ?>'&LOW_ (Oz) ǯų>?5&+ȊȖ
REi+õ=ʎ6 DNA z~] (Pol) α<Oz *ĺ>;/ Pol βPol γPol εPol ηPol

IVKlenow fragment exonuclease- (KF exo-) *Ëʑ>'ƛ<')"%, G:C-C:G fZmY+ǻǯ*ʫ>REiʢçƈ¿&>'Ⱥ

=Oz
<?>

ƕOz ¥ğ*9 G:C-C:G fZmY+ïČ')>÷ȿŪ>REiʢçƈ¿'%8-oxoG +<)>ʢç+ǯųǥ&>REiYloafG(Gh) '+ǵŪ­+G|lEfG (Ia) %ZqG|lYoafG(Sp) Ȉ<?%>KF exo-,?<+ƈ¿*ĺ%Eei'REiBõ=ʎ6

+õ=ʎ6ãǪ, Gh/Ia +ƕ Sp ;=9ˏ
1

'ƛ<

')"%

>5Oz*ĺ>REi+õ=ʎ6: Oz B=ʄ
'ƛ<

')"%

BɰĽ:©ʨãǪ, Gh/Ia +ęù'ǂʊ>'ɍ=Oz +ƕ Gh/Ia : Sp ;=9 G:C-C:G fZmY'ȍĄ?%>&ƨȊȖ&, G:C-C:G fZmY+ǻǯƸƵ+ɥƛ*¿'% Oz *ȅȀȜ 2 Ț'Ȝ 3 Ț&,ɨȠȐĪBǰ

%Oz

%ʣɝ)ƈ
;/ Gh/IaSp'REi+Ěėĺ+ĭİŪ:?<+ĚėĺBü7ƨʧ DNA +ƽ6Ŏù
DNA zDNA ùųãǪ~]*;>REi+õ=ʎ6ãǪ:ƈ¿=ʄ

Oz > Gh/Ia > Sp ')"¥Ü+ıˋȱƭ*#BȠÔ%ȺĹ+ȱƭ2 ƨ+ǄȫȱùBƤ> Oz:G Ěėĺ,3 ƨ+ǄȫȱùBƤ> Ia:G : Sp:G Ěėĺ;=9ĭİ&>'ȍĄ??,Ěėĺ+ĭİŪǄȫȱù+Ɛ*²ĩ%>8&>'<?ƕsp3 ǝȫB9# Gh/Ia : Sp *ĺ% Oz , sp3 ǝȫB9ƨʧȺ

DNA Bƽ8*ĢǠ+ DNA +Ƶʓ'ª%>'ǖ?#5=+ȱƭ Gh/Ia : Sp Bü7ƈ¿ DNA *ǂ2>'Oz Bü7ƈ¿ DNA *]+.":ƈ¿=ʄ=Ŀ)ıʳ+ DNA z
DNA ùų+ãǪɍ

> DNA z~~]óŦ*>Ěė+õ=ʎ6<?>ɧ>'DNA ɜɛ*

"'Ⱥ

Ƈ

> Oz *ĺ>REi+õ=ʎ6+Ŗʿ,Ŀ)+ȱƭɲ"ŬĘ+55ɜɛȲȳ?ƣȮǼ*¢+ƈ¿*ǂ2>' G:C-C:G fZmYBǯ:)>+8Oz Bü7ƈ¿ DNA (+;)ɜɛʘȒBȰ>+

Bƛ<

*>

'Ťɝ'?>Üʐ;*?5&* Pol α;/ Pol βPol γPol εPol ηPol IVKF exo-

 Oz *ĺ%õ=ʎ7Ěė*# %,ɥƫ?%+ɜɛƸƵ: DNA ùų*

> Oz +Ŗʿ*#

4 Ț&,DNA ɜɛ+ţǼŗÝBż
ùų*ʫ> DNA z~]+Oz Bü7ƈ¿ DNA%,Ʀɥƛ)ʟÕ9Ġ

DNA z

~]

&Ȝ

;/ƈ¿=ʄ

DNA!Oz *ʫ%Ʀɥƫ&" Pol δPol ζPol ιPol κREV1 + Oz *ĺ>ƈ¿=ʄóŦ;/Ěėõ=ʎ6óŦBɥƫ5Ȝ 5 Ț&,?5&* Oz *ĺ>Ěė+õ=ʎ6Bɥƫ DNA z
]+!REV1 ¥ğ+ DNA z

Oz:G Ěėĺ+ĭİŪ*ķ%~~]Ëʑ%REiBõ=ʎ7+,

>'Ⱥ

Oz:G
2

;/ Oz:AOz:COz:T Ěėĺ+Ěėĺ+ĭİŪB DNA z~]óŦ'ǤĞŪıˋ*;=ɥƫ<*Ȝ 6

Ț&,REiʔȳʠÖREië­;=9<*ʢç?:
KF exo-;/ Pol αPol βPol ζPol ηPol ιPol κREV1 Bǰĺ> DNA z% Oz ʔȳʠÖ*~]óŦBɥƫ+ȱƭȜ 4 Ț;/Ȝ 6 Ț+ȊȖȱƭ<Oz *ĺ>R;/¥Ü+ȊȖĘþ

Ei+õ=ʎ6Oz ë­+ęù, REV1 BʰÉ%+ DNA z=Oz ʔȳʠÖ+ęù, REV1 ' KF exo-Bʰ DNA z%>'ƛ<

'*ȅȀ

*%~]*Ëʑ%Ȝ 5 Ț&, Oz REi'ƣ9ĭİ)Ěė

')"¥*ß

ĺBŕų&>'Bƛ<~]*Ëʑ=DNA z~]*;>Ěė+õ=ʎ6ɥƫ+ȱƭ''9*Oz  G:C-C:G fZmY+ïČ')>REiʢçƈ¿&>%REiʢçƈ¿&> Oz *ĺ%XfX

>'BœȍĄ%

BÂÅǼ*õ=ʎ7 DNA z%Oz ʼŋ*œ~], REV1 +6&>'ĞǵïŪBƤ%ʔȳʠÖ'%ǯųęùĠ+ DNA zȣ)ƈ¿')=ŝ>'ƛ<ƕPol ζ , Oz ë­) Oz ʔȳʠÖ&>;>'ƛ<<DNA ɜɛʘȒ*')"<*Oz ~]*;> DNA ùųʭĴ>ʣ')"&)¢+ DNA zãǪɍ=ʄ

~]*;> DNA ùųBʭĴ

% DNA Bùų&>'ƛ<"+ȱƭ;=Oz ʔȳʠÖǯųęù&9Pol ζ ')Qf?>'&Oz ʔȳʠÖ*;> DNA ɜɛ+¼ƻBċʞ&>÷ȿŪ>+&,)%>5REV1 , Oz *ĺ%XfXBÂÅǼ*õ=ʎ7ą+ DNA z'Ⱥ~]&=+ REV1 *;> Oz *ĺ>XfX+ÂÅǼ)õ=ʎ6*;=Oz<ǯ> G:C-C:G fZmY+ǻǯBċʞ&>'ȍĄ?Ś,ƶ)ȭȾBǰ
ǯ­Í*


% Oz

<ǯ>ȘǠĞǵ+ZwQfBɥƫ>'&> Oz +ȘǠĞǵɰǻȿ*# %ɮȭ*ɥƫ>'Ťɝ')>@

%ŝ<?ȱƭ'ƨȊȖB,8' in vitro &+ȊȖȱƭBȴù>'&ƣȮǼ* Oz<ǯ>ǞȘǠĞǵ+ǻǯƸƵ+ɥƛ*#)>'Ⱥ

3

<?>

Ǵɱɢ
8-oxoG8-oxo-7,8-dihydroguanine (8 LO^REi)2-ME

2-mercaptoethanol

BRCT

BRCA1 C-terminal

BSA

bovine serum albumin

CPD

cyclobutane pyrimidine dimer (XQs`ēqctcalf thymus

DFT

density functional theory (ĸŎǆʫƐǭɶ)Ghguanidinohydantoin (REiYloafG)

h

human

Ia

iminoallantoin (G|lEfG)Iz

2,5-diamino-4H-imidazol-4-one (G|a_)

KF exo-Klenow fragment exonuclease-Oz

2,2,4-triamino-5(2H)-oxazolone (LOW_)

PMSF

phenylmethylsulfonyl fluoridePol

DNA polymerase (DNA z

Purpurine (tPyrpyrimidine (qSCRF

self-consistent reaction field (ɈŊǟƉȅóŦę)Sp

spiroiminodihydantoin (ZqG|loafG)THF

tetrahydrofuran (dfogr)Tmmelting temperature (ɔɥǕŎ)XP

xeroderma pigmentosum (ɎȫŪǾǹ)

y

yeast

|YaG{)

~])

)
|Y)

4

Ȝ1Ț
ōɶ
DNA ',ʝ¨ŬĘ+Ȳŵ'ǻǫBżˏÕĨǯ­ǥɾ&=+ DNA +ɜɛ*;"%ĨīBƾ'&>úƝ*DNA ,­Íğơ?%?)<+ƶ)ʢçZfZ*ŋ*=çĪǥɾ&> DNA ,ʢç?ƈ¿Bö>+ƈ¿ DNA ·ş"ęùɜɛʘȒ*%ɲ"ŬĘɜɛ?ȘǠĞǵBőʂ'>+ȘǠĞǵ,C:ȹçB,8ƶ)ʝ¨Ǹ+őʥ')>
DNA Ěė+1)!REi,¢+Ěė;=9ʢçʹ«¬ƣ9ʢçBö:%ǯųREiʢçƈ¿,ɜɛʘȒ&Ȃɚʧ½*Eei:REiõ=ʎ5?ęùƣȮǼ*REiG:C-T:A fZmY

<b|:XfX1+ǞȘǠĞǵ&>;/G:C-C:G fZmYǯ> (Fig.1)Ǧ*?<+ȘǠĞǵÂÅǼ*ǯ>ʢçƩ¦'%ƘÆǢĻp`| B2 (p) :p`| K3 (~hYL) ĩđ*sĩđ*yr

>ȬğȵǢĻγ ȵǢĻ~b

>÷ɠÆǢĻFe2++Ǔß:ʘʢçǄȫ+ǓßwLOXYMBǻǯ>ɬɓ+ǓßJgwLOXg+ǓßYLO\`+Ǔß)(<?> 2)5cf*%,öäĈǡ*;"% G:C-C:G fZmYǯ>'Ęþ?%> 3)REi+fZmY*;>ǞȘǠĞǵ,ƶ)ʝ¨Ĩ&ƳÔ?%>°'%CŶÛʝ¨Ĩ&> p53 ʝ¨Ĩ+ CpG W

Gf:Cʝ¨Ĩ&> K-ras ʝ¨Ĩ+Ug 1213 &ˏ˅Ŏ*ƳÔ?%1) 3-6)Ǧ* K-ras ʝ¨Ĩ*#> (Table%,ǞȘǠĞǵ*;"% GTPase ǍŪ¬ȭȾ

Ĝƿ'ǯĩ*ʫA>ǎ+XRhȰʆ+ſȳǼ)ǍŪçſȳ?ɃǺç`nQ'%®ǰ>'Ɨ*ƛ<')"%>+8?<+ǞȘǠĞǵ*#%ȊȖ>',ǞȘǠĞǵ*;=ǯ>C+ǻǹ~Mi[}+ɥƛ*
&>

5

%ʣɝ

Fig.1 The mutations caused from oxidatively damaged guanine.

Table 1 Type of genetic mutation in K-ras gene and its frequency in type of cancer 6).
mutation typecodon 12codon 13colon cancer

pancreatic cancer

lung cancer

CGT (Arg)

1.1%

12.2%

2.4%

GCT (Ala)

6.3%

2.3%

6.3%

TGT (Cys)

8.5%

3.3%

40.7%

GTT (Val)

22.6%

29.7%

19.8%

CGC (Arg)

0.5%

0.1%

3.1%

GCC (Ala)

0.2%

0.0%

0.1%

TGC (Cys)

0.5%

0.1%

3.1%

GTC (Val)

0.1%

0.1%

0.1%

GGT (Gly)GGC (Gly)

6

8 LO^REi (8-oxo-7,8-dihydroguanineː8-oxoG) ,ƶ)Ʃ¦&ǯų>¤ɘǼ)REiʢçƈ¿&= (Scheme 1)ƣ9ȊȖʕC&*ĺ>Ěė+õ=ʎ6ıˋ:ȭȾÍ*Ƶʓɥƫ<ıʳ*ŕų> 8-oxoG:A>?5&*8-oxoG&) 7)ȱƟ

>ĞǵZwQfɥƫ;/ 8-oxoG:C Ěėĺ+Ƶʓ9ƛ<')"%> (Fig.2) 8-10)+88-oxoG , G:C-T:A fZmYBőʂƈ¿&
>'Ⱥ

<?%

>ƕ8-oxoG ,REi',ĚėĺBŕų&Ȃɚʧ½*
ıʳ* K-ras ʝ¨Ĩ&6<?>;

REiƂÈ?)
Yǯ%>*9*G:C-C:G fZm

A< (Table 1) 3-6)8-oxoG &,+ǻǯBɳƛ&)#5=G:C-C:G fZmY,¢+REiʢçƈ¿*;"%őʂ
?>'Ⱥ

<?>

O
N

NH

N
dR

N

O

H
N

NH

O
N
dR

NH 2

G

O
HN

N

NH 2

HN
O

N

N

HN
dR

O

H
N
O
N
dR

Iz

O
∗

dR O

Sp

NH 2

NH

N
H

N

∗

N

8-oxoG

NH 2

NH 2

NH 2

O

O

N

∗

N
dR

N
H

Ia

Gh
+H2O

H 2N

NH 2

N

O

HN
dR

O

Oz (closed-ring)

NH
N
dR N
H

NH 2
COOH

Oz (open-ring)

Scheme 1 Oxidation products of guanine. Asterisk indicates the sp3 carbon.
7

NH 2

H
N

H 2N

O

N
dR

N

N H

H
H N

O

N
N

N
N

O

H
N

O

dR

8-oxoG (syn) : A (anti)

N

NH

N
dR

H
H N

N

N

N H
H

O

dR

8-oxoG (anti) : C (anti)

Fig.2 The structures of 8-oxoG:A and 8-oxoG:C base pair 8-10).

REi+ʢç&) 8-oxoG +<)>ʢç*;"%9ǯų>ƈ¿'%G> (Scheme 1) 11-13)Iz |a_ (2,5-diamino-4H-imidazol-4-oneːIz) Ȉ<?%ƨʧ;/ƨʧ DNA +REi+Æʢç*;=ǯų>' 13-15)%Iz ,in vitro;/ in vivo *%* G:C-C:G fZmYBőʂ'ƛ< *)"% > 16, 17) Iz , pH 737 ºC Ʃ¦&,éǔƥ 147 Õ&ś*
LOW_ (2,2,4-triamino-5(2H)-oxazoloneːOz) 1'ßǄÕɥ?> (Scheme 1) 11)
ıʳ*Ȼɇ+ DNA Wt <REi 107 ¸* 26 ÕĨ+ Oz ƳÔ?%> 18)5ƣʏ+Ęþ&,5-~bXfXĩđ&+Æʢç*;= p53 ʝ¨Ĩ
+ CpG WGf*

> Oz +ǯųʤƤŮ*Ĝß>'ƛ<')"% > 19)¥;=Oz +ǯǥĪǼ)Ŗʿ, Iz *ǂ2>'ġǟɠ&)?5&+ȊȖ

<Oz *ĺ>REi+õ=ʎ6 DNA zpolymeraseːPol) α

'Ⱥ

<?>

~] (DNA;/ Pol βPol γPol εPol ηPol IVKlenow fragment exonuclease-(KF exo-) *Ëʑ>'ƛ<')"%= 20, 21)Oz , G:C-C:G fZmY+ǻǯ*ʫ>REiʢçƈ¿&>'Ⱥ<?>ƕOz ¥ğ*9 G:C-C:G fZmY+ǻǯ*ʫ>÷ȿŪ>REiʢçƈ¿'%REiYloafG (guanidinohydantoinːGh) 'ZqG|
lYoafG (spiroiminodihydantoinːSp) Ȉ<?%¦Sp ,ĚėŪƩ¦*> (Scheme 1)Gh ,ʢŪƩ

> 8-oxoG +<)>ʢç+ǯųǥ&> 12, 22-24)5

Gh +ǵŪ­'%G|lEfG (iminoallantoinːIa) Ȉ<?%>

(Scheme 1) 25)DNA z>~]óŦ*%(!<+ǵŪ­*Ōɗ»"%
8

Ʀ

ƛ<

')"%

?<+ƈ¿*ĺ% KF exo-Eei'REiBõ=)ʎ7'Ęþ?%=+õ=ʎ6ãǪ, Sp ;=9 Gh/Ia +ƕˏ> 26)¥*ß')"%©ʨãǪ, Gh/Ia ;=9ɍOz *ĺ>REi+õ=ʎ6
'ƛ<

')"%

'9ƛ<

;/ Oz B=ʄ> 20)#5=G:C-C:G f

ZmY+ǻǯ1+ķŎ, Gh/Ia : Sp ;=9 Oz +ƕˏ

'ȍĄ?%>&ƨȊȖ&, G:C-C:G fZmY+ǻǯƸƵ+ɥƛ*>ƈ¿'% Oz *ȅȀȜ 2 Ț'Ȝ 3 Ț&,ɨȠȐĪBǰ
Oz : Gh/IaSp *ĺ> DNA z*?5&* Pol αÜʐ; Oz *ĺ%õ=ʎ7Ěė*#z%Üʐ¥Ü+~]*;>REi+õ=ʎ6ãǪ:ƈ¿=DNA ùųãǪ+ıˋȱƭ 20, 26) *#

ʄ

%ʣɝ')

%ȺĹ

;/ Pol βPol γPol εPol ηPol IVKF exo-%,ɥƫ?%DNA ɜɛƸƵ, DNA~]B,8+¢ƶ)`nQɾʫ>ʼŋ*ɜʶ)ƸƵ&=

DNA ùųBɦħ> DNA z~]*#%,ġɄɐ&, 5 ȓˈof&, 15 ȓˈ9Ƥ%>+8Oz Bü7ƈ¿ DNA +ɜɛƸƵ: DNA ùų*+Ŗʿ*#%,Ʀɥƛ)ʟÕ9Ġ

DNA *ĺ> DNA z&Ȝ 4 Ț~]óŦBɥƫ

9

> Oz

<Ȝ 6 Ț&, Oz Bü7ƈ¿Ȝ2Ț
REiʢçƈ¿ːREiĚėĺ+ĭİçJkP+ɨȠ(ƨȚ+ÍĶ*ʫ%, Molecules, 17, 6705-6715 (2012) *ǻɘ6)

Ȝ 1 ȡ Ƚƞ
Ȝ 1 Ț+ōɶ&9ʐ2;*REi,¢+Ěė*ǂ2%ʢç?:ɜɛ

ʘȒ*%ʢç?REi*ĺ%Eei:REiõ=ʎ5?>'

G:C-T:A;/ G:C-C:G fZmYǯ>ƨȔ&,8-oxoG &,ɳƛ&

G:C-C:G fZmY+ǻǯƸƵ+ɥƛBȀǼ'%)ZmYBőʂ÷ȿŪ+>ƈ¿'% Oz?> (Scheme 1)?5&+ȊȖ

;/ Gh/IaSp <<Gh/Ia : Sp *ĺ>REi+õ=ʎ6*ǂ2

Oz *ĺ>REi+õ=ʎ6ãǪ;/+Ś+©ʨãǪˏ

'ƛ<

')

> 20, 26)"%&DNA ƈ¿*ĺ>Ěė+õ=ʎ6óŦ+ƸƵ*#]*;>Ěė+õ=ʎ6ʘȒ*ɀĚėƈ¿*ĺ%DNA z)"%%ʐ2>DNA z~%õ=ʎ5?>ĚėʦēĚė'ĚėĺBŕ

DNA Ěė'ȥʧ+ N-R

ų>',ʣɝ&>

UXgȱùʩə%ǯ

~]EeiBÂÅǼ*õ=ʎ7ƛ<'=+ǫɺ, A-rule 'ā-?% > 27)5ɀĚėƈ¿+||cQ­&>dfogr (tetrahydrofuranːTHF) *ĺ%Ěė&,)?>'ƛ<')"%'ɀĚėƈ¿*ĺ%Eeiõ=ʎ5?>'>+ɳƛBñȺ*>ǫɺ,Eeiƣ9ȗʪʛ'*)>REi,ɀĚėƈ¿*ĺ%õ=ʎ5?)¿*ĺ%REiõ=ʎ5?>'
ʎ6+ʣɝ)ČĨ')"%>'+ʽȕBƤ> (ȗʪʛùŪ) <THF *ĺ%qõ=ʎ5?'ɳƛ?%

ùŪ>'qõ=ʎ5> (Fig.3) 28)THF 'q,ǄȫȱùBŕų&)qʑŋ+ G:C Ěėĺ: A:T Ěėĺ'ú<'>+ G:C-C:G f+&ƈǫɺ,REi'+Ǆȫȱù+ŕųõ=> 20)
10

OH

HO
O

O

OH

HO

Pyrene : THF
Fig.3 The structures of Pyrene:THF base pair 28).

&ƨȚ&, Oz:GDNA z;/ Ia:GGh:GSp:G Ěėĺ+ĭİŪBǂʊ>'&
%REi+ƂÈãǪ:+Ś+©ʨãǪ Oz > Gh/Ia >~]óŦ*Sp ')"¥Ü+ıˋȱƭ 20, 26) Bɳƛ>'Bɬ6¥Ü*ʤĨçĪɨȠ*;"% Oz:G Ěėĺ, 2 ƨ+Ǆȫȱù9"ŌʽƵʓ&>'ǖ?%ƕIa:G ' Sp:G *#P*#%9ŕų>Ěėĺǖ?%%,ȠÔ?%> 20)

>?<+ĭİçJk

< 2)Oz:G Ěėĺ'+ĭİŪ9ǂʊ?%)5

Gh:G Ěėĺ, Beckman <*;"%ǖ?% > 29)ƨȚ&,ĸŎǆʫƐǭɶ
(density functional theoryːDFT)
SCRF) Bǰ%Ia:G;/ɈŊǟƉȅóŦę (self-consistent reaction fieldː

;/ Sp:GGh:G Ěėĺ+ĭİçJkPBȠÔOz:G

Ěėĺ+ĭİçJkP'ǂʊ

11

Ȝ 2 ȡ ƕǈɨȠƝʪȉȶ+8ʢçƈ¿'REi+ĚėʟÕ2'-eLOX+ǝȫ' C1'«+Ǆȫ 1 #BʰyZ+ C1'«%É%+ïĨBʰ+Ś C1'~b*ǄȫïĨB 2#£>'&N-~bçưʢĚėĺ'%Gaussian 03 30) +ƣʛçtR}Bǰ
Bǰ%B3LYP/6-31G**v&ôƒƵʓBƣʛçƵʓ'ƣʛçƵʓ%Ȅȗ+ĭİçJkPBɨȠ<*Ǆ+ĭİçJkPBȠÔ>8*Onsager reaction field ǈ (ε = 78.39) BǰP,Ő (1) BǰĚėĺŕų*>Jk%İȸ

ΔE = E(base pair complex ‘X:Y’)-[E(isolated base ‘X’)+E(isolated base ‘Y’)]Ȅȗ+ƣʛçƵʓ, GaussView Bǰ%÷ɠçFig.4, 6, 8, 9 *ȍ

12

(1)

Ȝ 3 ȡ ȱƭ'ȺĹ
3-1. Ia:G Ěėĺ+Ƶʓ'ĭİçJkP+ȠÔ
Ia ,REi' 3 ƨ+Ǆȫȱù*;=ĚėĺBŕų&>' kino <*;=ǖ> 2)ƨȚ&,+ 3 ƨ+ǄȫȱùBŕų)< Ia '=ŝ>÷ȿŪ+>?%

8 #+UrK~X (Ia1-Ia8) 'REi+ĚėĺB B3LYP/6-31G**v&ǅ8
Ia1:G-Ia8:G ??+ƣʛçƵʓ, Fig.4 *5?<+ Ia:G Ěėĺ??
%ȠÔĭİçJkP, Table 2 *ȍIa1:G +ĭİçJkP,Ȅ

*#ȗ&, 29.5 kcal/mol &=úƶ*ȠÔf^QİçJkP (30.9 kcal/mol) 'ʼŋ*ʏ ºĺ+ƕ 3 ƨ+ǄȫȱùBŕų>'"?,Ia:G Ěėĺ' C:G Ěė<Ⱥ>'ˌ2'&,)?

Ia3:G : Ia4:GIa5:GIa7:GIa8:G +ĭİçJkP95 29.5 kcal/mol &*ß=Ǆȫȱù*ʫ)%cQē+ C:G Ěėĺ+ĭʟÕ+ĭİçJkP*>Ŗʿ,ľ

'Bȍ>ƕ&Ia2:G ' Ia6:G ,ÕĨÍǄȫȱù (O4-H8) (Fig.4B) BƤ%

JkP, 28.7 kcal/mol=+ĭİç

"+#+Ěėĺ, O4 «+ʹĨĸŎ+ʼŁđç*;"%¢+ Ia:G Ěėĺ;=9ĭİç>'Ⱥ<?>#5=REi'+Ǆȫȱù*ķ>ʹĨĸŎ+¬*;"%REi'ŕųĚėĺ+ĭİç,¬>'ŨA?>
?< 8 #+ Ia:G Ěėĺ+Ǆ*

>ĭİçJkPB SCRF º'%ȠÔ

+ȱƭIa1:G +ĭİçJkP 24.1 kcal/mol 'ƣ9ˏƺ* 24.0 kcal/mol 'Ia5:G ˏ,S­

"Ia1 , (+)-S ʠȷB9!Ia5 , ()-R ʠȷ&> 31)Ia1 ' Ia5 +ʙR­+ʙ',ˌ'&,)
Ǆ*

&>+&Ia5:G ' Ia1:G 3'C(úĭİŪ&>



> Ia3:G ' Ia4:GIa7:GIa8:G +ĭİŪ, Ia1:G : Ia5:G ;=9¬Onsager reaction field ǈ*
ǄÕĨ&Ď5?%"%,ǘɾ,Ǭǧ+OpdF*ʠȷ?+ÿ=,>%OpdF+éŘġ'OpdF<ǄBʰð>8*;=Ġ+JkPŤɝ'?>#5=ȠÔ?>JkP,
13

OpdF+ġ*²ĩ%>+&OpdFľȒĚėĺ+ĭİŪĜ 32)S ­+ƵʓBǂʊ>'OpdFéŘ+ġ, Ia3 > Ia4 > Ia1ƕR ­&, Ia7 > Ia8 > Ia5 &"?ƎIa1:G
İ&>?<+ɨȠȱƭ,Ǆ*>'BȍĄ%

Table

2;/ Ia5:G Ěėĺ¢+Ěėĺ;=9ĭ

>ĭİç,Ěėĺ+ÕĨWG[*²ĩ>Stabilizationenergies(kcal/mol)

of

base

pairs,

obtained

from

the

B3LYP/6-31G**-optimized geometriesa. Reproduced from Suzuki M. et al., Molecules, 17,6705-6715 (2012) with permission from MDPI.
Base pairΔ EDFT

Δ ESCRF

Base pairΔ EDFT

Δ ESCRF

Ia1:G

29.5

24.1

Gh7:G

19.9

19.5

Ia2:G

28.7

19.3

Gh8:G

19.8

16.9

Ia3:G

29.5

17.0

Gh9:G

21.0

19.1

Ia4:G

29.5

23.5

Gh10:G

20.9

17.3

Ia5:G

29.5

24.0

Gh11:G

20.6

20.6

Ia6:G

28.7

19.7

Gh12:G

20.4

19.6

Ia7:G

29.5

18.1

Gh13:G

20.3

16.6

Ia8:G

29.5

22.6

Gh14:G

20.5

17.1

Gh1:G

21.0

18.9

Gh15:G

20.8

18.5

Gh2:G

20.9

16.7

Gh16:G

21.1

19.0

Gh3:G

20.5

21.4

Sp1:G

28.2

18.8

Gh4:G

20.4

19.5

Sp2:G

28.2

19.9

Gh5:G

20.4

18.2

Oz:G

20.7

16.3

Gh6:G

20.5

16.8

a

ΔEDFT, in vacuo; ΔESCRF, SCRF = Dipole, dielectric = 78.39, in water
14

A

B
H
O
H 2N

HN
∗
N
dR

H
N H

O

N

N H N
N
O

N H

N

HN 1 2

dR

N6 5

dR

O

H N
H

7

4

8

N H
H

3

N

O

N
N

H N
N

O

dR

H N
H

C

Ia1:G

Ia2:G

Ia3:G

Ia4:G

Ia5:G

Ia6:G

Ia7:G

Ia8:G

Fig.4 The Ia:G base pairs. (A) The proposed Ia:G base pair. Asterisk indicates the sp3 carbon.
(B) The proposed Ia:G base pairs and the associated hydrogen bonds. This numbering is thesame as used in reference 25. (C) The geometries of Ia1:G-Ia8:G optimized by ab initio
calculation. The stabilization energies are shown in Table 2. Reproduced from Suzuki M. et al.,
Molecules, 17, 6705-6715 (2012) with permission from MDPI.

15

3-2. Gh:G Ěėĺ+Ƶʓ'ĭİçJkP+ȠÔ
Beckman <, Gh:G Ěėĺ, 2 ƨ+ǄȫȱùBŕų>'ǖ%&,YSf­Jl­ (Fig.5A) ;=9ĭİċʈʉ'OţB 1 #9"%'ƅİ?%= 29)Ǆǘǐ> 33)Gh , 3 #+>+& (Fig.5B)Beckman <+ǖ Gh:G ĚėĺBŕųŝ>Ƶʓ, 16 ʑ= (Gh1-Gh16) Ⱥ<?>?<+ Gh1:G-Gh16:G Ěėĺ+ƵʓBǅ8+ŚĭİçJkPBȠÔ%ǂʊ (Fig.6Table 2)Gh1-Gh16!Gh1-Gh8 , ()-S ʠȷGh9-Gh16 , (+)-R ʠȷ&>Ȅȗ*+

Gh:G Ěėĺ+ĭİçJkP,2 kcal/molǄ*%, Gh3:G ƣ9ˏ



ŉ)

"?<+ȱƭ,ɪ+ 3-1&ʐ2OpdF+éŘ+ŉUrK~X+ʙ

;=9ĭİŪ*ġ)Ŗ

>Ŝ"%S ­+ Gh:G Ěėĺ+&ƣ9ľFéŘ&> Gh3:G ƣ9ĭİOpd"ƕ Gh11:G , R ­+&ƣ9ĭİ&=+ĭİçJkP, 20.6 kcal/mol"Gh3 ' Gh11 +ʙ

=?<,ƣ9ĭİ)ǵŪ­Bȍ%

'BȍĄ%ǵŪ­&>+&?<+ș­ǵŪ­,ǄǘǐÍ*Ëĩ%¥ÜAller <, DNA ƨʧ*,S­=ˌ'&,)

ƕ R ­;=9REi+õ=ʎ6ãǪɍĆ%"?*ĺ%

ĭİçJkPBȍ21.4 kcal/mol &"S­+ƣ9ĭİ)Ěėĺ, 16.8 kcal/mol + Gh6:G

ʿBƤ>'Bȍ%

>É%+

R­

¥

&

<S ­+>S ­' R ­,Ğ>'Ⱥ<?> 12, 34)% Gh + S ­+ƕʦē'%;=ɍ

'BƆ= 34)?<+ɨȠȱƭ, Aller <+ıˋȱƭBɳƛ&>9+&"

5Ȅȗ'Ǆ+ƕ*

> Gh:G +ĭİçJkP, Ia:G ;=9¬"ǖ?> Ia:G Ěėĺ,ƨ+ǄȫȱùBƤ%>'ȫȱù+Ɛ*²ĩ>'ŨA?>?<+'<Gh/Ia *ĺ%REiƂÈ?>ʳ,Gh , Ia *Ōɗ»"%

>

9?)

16



<ĭİçJkP,Ǆ

A
O

N
HO

∗

N
dR

H
N

NH

N
H

O

O

∗

N
dR

NH 2

enol-1

N
H

OH

N

NH

O

∗

N
dR

NH 2

NH

N
H

NH 2

enol-2diketo

B
H
N
O
N

O

N

H

∗
N
H

dR

NH 2

Fig.5 The structure of Gh. (A) Neutral Tautomers of Gh shown by Verdolino et al.33) The diketo
form showed only one form in here, which used Gh:G base pair proposed by Beckman et al.29)
The enol tautomers showed three forms having the same guanidinium group as the diketo form
used in Gh:G base pair. (B) The curly arrows indicate the rotation axis. Asterisk indicates the sp3
carbon and chiral center.

A

H
N O

H 2N
HN

∗
N
dR

       

N H
O

O

N
N

H N
N

dR

H 2N

B

Gh1:G

Gh2:G
Fig.6 Cont.
17

Gh3:G

Fig.6 Cont.

Gh4:G

Gh5:G

Gh6:G

Gh7:G

Gh8:G

Gh9:G

Gh10:G

Gh11:G

Gh12:G

Gh13:G

Gh14:G

Gh15:G

Gh16:G
Fig.6 The Gh:G base pairs. (A) The proposed Gh:G base pair. (B) The geometries of Gh1:G-Gh16:G optimized by ab initio calculation. The stabilization energies are shown in Table 2.
Reproduced from Suzuki M. et al., Molecules, 17, 6705-6715 (2012) with permission from
MDPI.
18

3-3. Sp:G Ěėĺ+Ƶʓ'ĭİçJkP+ȠÔ
Sp 'REi, Ia:G Ěėĺ'ˈª 3 ƨ+Ǆȫȱù*;=Sp:G ĚėĺBŕų> 2)5Ǆǘǐ*>'ǖ?%
ƣ9ĭİ%>'ƅİ?%%,Sp +fSfĞǵŪ­ (Fig.7) > 33)Sp + 2 #+ș­ǵŪ­&> Sp1 ' Sp2 *#%,Sp1 ()-S ʠȷSp2  (+)-R ʠȷ&>Sp1:G ' Sp2:G +Ȅȗ*>ĭİçJkP,Ë* 28.2 kcal/molǄ&, Sp1:G  18.8 kcal/molSp2:G  19.9
kcal/mol" (Fig.8Table 2)3-1 &ʐ2OpdFéŘ+ʙ

, Sp1:G ;=9OpdFéŘľ
5Ȅȗ'Ǆ+ƕ*BȺű>'Sp2:G

8Sp2:G +ƕĭİ&>'Ⱥ<?>%Sp:G Ěėĺ+ĭİçJkP, Ia:G Ěėĺ+&ƣ9ĭİ&" Ia1:G Ěėĺ*ǂ2>'¬"Kornyushyna <+Ęþ&,

Sp *ĺ>REi+õ=ʎ6ãǪ, Gh/Ia ;=9¬ƈ¿=ʄ
Gh/Ia *ǂ2 Sp ,œscQ?% 26)¥B05

ȱƭ, Kornyushyna <+ıˋȱƭ'İŪǼ*Ɋ%19>

DNA ùų9>'&ŝ<?ɨȠ

O
HN
N
HO

N

O
HN

NH 2
HN

N

∗

N

O

dR O

imine enol-1

N

N
N

∗

NH 2
N

dR O

O

triketoimine enol-2

O

N

∗

dR O

NH 2

N

HN
O

∗

OH
HN

NH 2

N

dR OH

imine enol-3

Fig.7 Neutral Tautomers of Sp shown by Verdolino et al.33) The diketo form showed only one
form in here, which can form Sp:G base pair proposed by Kino et al.2) The imine enol tautomers
showed two forms, and the position of their imine enol is at hydantoin ring. Asterisk indicatesthe sp3 carbon and chiral center.

A

B
H
O HN

HN
O

N
dR

N H
N

O

N
N

H N
N

O

dR

H N
H

Sp1:G

Sp2:G

Fig.8 The Sp:G base pairs. (A) The proposed Sp:G base pair. (B) The geometries of Sp1:G and
Sp2:G optimized by ab initio calculation. The stabilization energies are shown in Table 2.
Reproduced from Suzuki M. et al., Molecules, 17, 6705-6715 (2012) with permission from
MDPI.

20

3-4. Oz:G Ěėĺ+ĭİçJkP+ȠÔ' Ia:G
Oz:G Ěėĺ*#;/ Gh:GSp:G Ěėĺ'+ǂʊ> 20)ƌ8%?B%,Ɨ*ĭİçJkPǖ?%%ɨȠŚĭİçJkPBȠÔ+ȱƭȄȗ&, 20.3

ƨȊȖƕǈ*ė$kcal/molǄ&, 16.3 kcal/mol 'ºŝ<? (Fig.9Table 2)+ȱƭ,Ia:GĚėĺ 3 ƨ+Ǆȫȱù&>ƕOz:G Ěėĺ 2 ƨ+Ǆȫȱù&>8Ia:G
Ěėĺ;=ĭİ&>'Bȍ%>)<?5&+ıˋȱƭ*;>'ƈ¿=ʄ

DNA ùų+ãǪ, Oz >Gh/Ia > Sp &" 20, 26)?&,)ıˋȱƭ'ƨȚ*

>ɨȠȱƭ,Ȇȃ>



+

@

?,

<Oz:G ĚėĺŌʽƵʓ&>+*ĺ%Gh/Ia

Sp , sp3 ǝȫB9!ʼŌʽƵʓ&>'ʣɝ)ʙ

;/<?>DNA &>'Ⱥ>Ōʽ)Ěė+Z`cOR DNA ƨʧ+ĭİŪBĜœ>ƕsp3 ǝ*ȫB9#ʼŌʽ)Ěėĺ, DNA ƨʧ+ĭİçBőʂ°-Sp Bü7 DNAƨʧ+ɔɥǕŎ (melting temperatureːTm º) ,ĢǠ+ DNA *ǂ2 20 ºC Ȓ¬<Sp ,ƨʧ+ǤĭİŪBƤŮ*¬>'Ęþ?%z~]Bǰ'> 35)#5=DNAıˋȱƭ,ƈ¿Ěėĺ+Ǆȫȱù+Ɛ'Z`cORãƭBȺű>'*;"%ɳƛ>'&>ƨȚ&,2'-eLOX@yZʟB~bė*ȷƇeBǰ%REiʢ

çƈ¿+ĭİçJkPBɨȠ5C1'ǝȫ+«ȷBĐİ*ƵʓBǅ8?*ßıˋǼ*ȠÔ?f^Q

cQē C:G Ěėĺ,ĭİçJkP 21.0 kcal/mol &>+*ĺ% 36)DFT ɨȠ*;"%ȠÔęù, 25.0 kcal/mol&=ıˋº;=9 4.0 kcal/mol ˏ"+Ȇȃ, B3LYP/6-31G**v&+ɨȠ*;>9+&>C1'ǝȫ+«ȷ+Đİ'Z`cOR,ƛ<
3 Ț&ȺĹ>

21*ʣɝ&>+&Ȝ

A

B
H
N

H 2N
N

O
H
O

N
N

H N
N

dR NH

O

dR

H N
H

Fig.9 The Oz:G base pair. (A) The proposed Oz:G base pair. (B) The geometries of Oz:G
optimized by ab initio calculation. The stabilization energies are shown in Table 2. Reproduced
from Suzuki M. et al., Molecules, 17, 6705-6715 (2012) with permission from MDPI.

22

Ȝ 4 ȡ ȱɶ%Ia1:G-Ia8:G +&, Ia1:G  24.1 kcal/molGh1:G-Gh16:G +&,Ǆ*

Gh3:G  21.4 kcal/mol 'ƣ9ĭİ&=Ia ' Gh +Ōɗ­+&, Ia1:G ƣ9÷ȿ
Ū+>ĚėĺƵʓ&>#5=Gh/Ia +Ȃɚʧ½*REiõ=ʎ5?>ʳ,
Gh ;=9 Ia +ƕƤÚ'Ⱥ<?>5Sp:G Ěėĺ*ʫ%,Sp2:G Ěėĺ+ƕ Sp1:G Ěėĺ;=9A

*ĭİ&=+ĭİçJkP, 19.9 kcal/mol"Ia:G Ěėĺ+ĭİçJkP'ǂʊ>'Sp:G Ěėĺ+ƕ Ia:GĚėĺ;=9ĭİçJkP¬Sp:G Ěėĺ Ia:G Ěėĺ;=9ĭİ&>'ȍ?+ĭİŪ+ʙãǪɍ,Sp ;=9 Gh/Ia *ĺ>REi+õ=ʎ6+ıˋȱƭ 26) Bɳƛ&>'@5Oz:G Ěėĺ+ĭİçJk%, 16.3 kcal/mol &"Kino <: Kornyushyna <+ıˋȱƭ*;P,Ǆ*=Gh/Ia : Sp 'ǂʊ>' Oz *ĺ>REi+õ=ʎ6,ãǪǼ&>'ȍ> 20, 26)ƨȚ*?%

>ɨȠȱƭ, Oz:G Ěėĺ Ia:G : Gh:GSp:G Ěėĺ;=ĭİ&>'Bȍ#5=DNA z~]*;>Ěė+õ=ʎ6ɥƫ:©ʨóŦ+ɥƫȱƭBɳƛ>8*,ƈ¿ĚėBü7Ěėĺ+ĭİçJkP&)Z`cORãƭB,8'¢+ǥŪBȺű>'Ťɝ&>'Ⱥ<?>%?<BȺű>',ƨȚ*<+ DNA z'Ⱥ~]Bǰ

%ǯ Kino <: Kornyushynaıˋȱƭ'ƨɨȠȱƭ+ɊBɥǇ>+*ŗș#

<?>

23

Ȝ3Ț
REiʢçƈ¿ːREiĚėĺBü7 DNA ƨʧ+
ƽ6+ɨȠ(ƨȚ+ÍĶ*ʫ%, Molecules, 19, 11030-11044 (2014) *ǻɘ6)

Ȝ 1 ȡ ȽƞÜȚ&, G:C-C:G fZmYBőʂ÷ȿŪ+>REiʢçƈ¿&
>Oz;/ Gh/IaSp 'REi+Ěėĺ&> G:OzG:GhG:IaG:Sp Ěėĺ+ĭİçJkPBʤĨçĪɨȠ*;=ȠÔ+ȱƭĚėĺ+ĭİŪ,Ǆȫ=?<+Ěėĺ+ĭİŪ, G:Ia > G:Sp > G:Gh > G:Oz &>ȱù+Ɛ*²ĩ%'ǖ?)<Ȝ 1 Ț:Ȝ 2 Ț+Ƚƞ&ʐ2;~ıˋ*;=Oz *ĺ>REi+õ=ʎ6, Gh/Ia : Sp ;=9ãǪ]Bǰ

%9 Oz +ƕ Gh/Ia : Sp ;=9ãǪɍɕA?>'ɍ+Ś+©ʨóŦ*ƛ<*DNA z')"%

> 20, 26)#5=ÜȚ*+ıˋȱƭ 20, 26) Bɳƛ>',&%ıʳ+ DNA z%ȠÔĚėĺ+ĭİŪ)~]*;>Ěėõ=ʎ6óŦ*&,)ƈ¿Ěė*;> DNA ƨʧ+ƽ6Ŏù%,Ěėĺ+ĭİŪƨʧ+ƽ6*ʼŋ*ůöŪˏDNA ƨʧBƽ8>;
ɛ?*

'

A?%

~]+Ǚʁ+."<&> (Fig.10)ʘð+ȊȖ*ƨʧ,ĢǠ+f^Q'Ęþ?%~], DNA) DNA ƈ¿, DNA ɜ> 37, 38)))<DNA ƈ¿*;"%ƽ8<? DNA ƨ

ʧ,DNA ɜɛ+ʳ* DNA z+¬BŽ

:Z`cORãƭȞ+ƶ)ǥ

>ɌǼ*DNA ɜɛ*ʫ> DNA z

Ū*;>ŖʿBö%&,

=+ïČ')=©ʨãǪ%ʼŌʽ+ DNA ƈ¿Bü7 DNAcQĚėĺ+ DNA *ǂ2>'ĭİ

#ƽ6:



> 39)&ƨȚ&,+ʼŌʽ+ DNA ƈ¿ DNA ƨʧBƽ

8:ĭİ*)>' 39)%ɜɛʘȒ*%,+ DNA ƨʧ+ƽ6

DNA +ɜɛãǪ+¬BŽǞ*ȅȀ 37)Fig.11 *ȍ%>;*G:Oz Ěėĺ, sp3 ǝȫB9ŌʽƵʓ&>G:Ia ' G:Sp Ěėĺ, sp3 ǝȫB9#8ʼ
24

Fig.10 DNA synthesis by DNA polymerases. (A) DNA synthesis of undamaged DNA isefficient. (B) DNA synthesis of damaged DNA is inefficient.

25

A

B
O

N
N
dR

N H

H
O

H
N

NH

O

O

N
N
dR

N

N
N H

C
H

HN dR

O

N

NH O
N
N H N
∗
dR
N
NH 2
N H O
dR

N

H

H N

H

H N

H

N H N
N
N H

O

H

NH O
∗
N

NH

dR

O

Fig.11 The proposed hydrogen bonding of (A) G:Oz, (B) G:Ia, and (C) G:Sp base pairs. Asterisk
indicates the sp3 carbon.

ŌʽƵʓ&>#5=Ia : Sp , sp3 ǝȫʴƄĚėĺ+ŌʽŪBņ'&+ȱƭ'% Ia : Sp BƤ> DNA , Oz BƤ>ęù*ǂ2DNA +ƨʧƽ6<?>+8?<+ƈ¿ĚėBü7 DNA ƨʧ+ƽ6Ŏù:)>'Ⱥ+ʙ=ʄBȺű>'&DNA z~]*;>REi+õ=ʎ6ãǪ:ƈ¿DNA ùųãǪ Oz > Gh/Ia > Sp ')"¥Ü+ıˋȱƭ 20, 26) 'ÜȚ+ɨȠȱƭ+ȆȃBɥǇ&>'ȺĭİŪ+ɨȠȱƭ
=ƨȚ*

)

ÜȚ&9ʐ2G:Ia Ěėĺ' G:Gh Ěėĺ+<REi+õ=ʎ6ʘȒ*%, Ia *Ōɗ»>'ǖ%%, Ia +6BȺűƨȚ&,G:Oz 5, G:IaG:Sp ĚėĺBü7ƨʧ+ĭİŪ'ƽ6ŎùBȠÔ>'&Üʐ¥Ü+ DNA z~]Bǰ

ıˋȱƭ 20, 26) Bɳƛ

>'Bɬ6?<+ƈ¿ĚėBü7 DNA ƨʧ+ĭİŪ+ȠÔ*,hPol β-DNA e 40) Bǰ+ʙ;/ƽ6Ŏù

+ʙ%ÕĨÞĪɨȠ*;=ƵʓƣʛçŚ*??+ƈ¿ĚėĺBţ*ü7 3 #+ĚėĺBŸÔ DNA ƨʧeBǰ26Ȝ 2 ȡ ƕǈ
2-1. ÕĨeRƨȚ&,hPol β +ȱƟƵʓ (PDB entry: 1BPY) 40) Bǰ%G:X Ěėĺ (X = Oz 5

, S-IaR-IaS-SpR-Sp) B?? 1 #ü7 hPol β-DNA eB®ų?<+e+ƵȢƕǈ+ɮȭ*#ˋ&ǰ%ɪ*ȍ¥Ü+ DNA z~]Bǰı

VjQLbg&,ƈ¿+ 5'½ʴƄĚė T &=Oz +ƺ+ĚėL

õ=ʎ6&,+ T *ĺ% dATP õ=ʎ5?> 20, 21)&Ã+ hPol β-DNA eɜù­+Ěė&> dCTP B dATP *dCTP +dtf&> G B T *ȷƇdtf-tG{+ 3'Ƨț+ G:C Ěėĺ,Ȝ 2 Ț+ʤĨçĪɨȠ&ŝ<? 5 #+ G:X Ěėĺ*??ȷƇE|lʢ'¢+ DNA ĚėʠÖ,ȱƟƵʓ+e`Bǰ

%ƵȢe+ G:X Ěėĺ+6Đİ%Macromodel

9.0 + OPLS2005/water Bǰ%Ƶʓƣʛç

2-2. ab initio ɨȠ
Macromodel 9.0 Bǰ%ƣʛçŚ+Ƶʓ<G:X Ěėĺ;/ G:X Ěėĺ*ʴƄ

> 2 #+Ěėĺ%?<É%+Ěė+ C1'«+ǝȫ' C1'Ǆȫ 1 #¥ğ+É%+%C1'~b*ǄȫB 2 ##>'&N ~bçưʢĚėĺ'ïĨBʰ (Fig.12) 20)2-1 &ʐ2;*“A1T1” ;/“G2X2”“G3C3”' (Fig.12Fig.15)

%B3LYP/6-31G** v&“A1T1”+JkPBȠÔ>8*Gaussian 0330)

Bǰ<*Ǆ*

78.39) Bǰ

>JkP+ȠÔ*,Onsager reaction field ǈ (ε =“A1T1”'úƶ*“G3C3” ;/“A1T1 + G3C3”+JkP9ȠÔ%??+ĭİçJkP (ΔE1
İȸ

27

;/ΔE3ΔE1+3) ,Ő (2)–(4) Bǰ%ΔE1 = E(“A1T1” of G:X complex (X = C))(2)– E(“A1T1” of G:X complex (X = Oz, S-Ia, R-Ia, S-Sp or R-Sp))

ΔE3 = E(“G3C3” of G:X complex (X = C))(3)– E(“G3C3” of G:X complex (X = Oz, S-Ia, R-Ia, S-Sp or R-Sp))

ΔE1+3 = E(“A1T1+G3C3” of G:X complex (X = C))(4)– E(“A1T1+G3C3” of G:X complex (X = Oz, S-Ia, R-Ia, S-Sp or R-Sp))

Fig.12 An overview of calculating the destabilization energies of DNA duplexes. Each Pol
β-DNA complex containing a G:X (where X = C, Oz, S-Ia, R-Ia, S-Sp or R-Sp) base pair was
minimized. G:X and each base pair adjacent to G:X is delineated in Fig.15. A:T base pair on the
5'-side of X was designated “A1T1”, G:X base pair was designated “G2X2”, and G:C base pair on
the 3'-side of X was designated “G3C3”. The destabilization energies of “A1T1” (ΔE1), “G3C3”(ΔE3), and “A1T1 + G3C3” (ΔE1+3) were calculated ab initio as the parts common to each model
duplex; each G2X2 base pair was excluded from the calculations. Reproduced from Suzuki M. etal., Molecules, 19, 11030-11044 (2014) with permission from MDPI.
28

2-3. ƽ6Ŏù+ȠÔ?? 5 #+Ěė (A1T1G2G3C3) +ïĨ C5 (xC5, yC5, zC5)N1 (xN1, yN1, zN1)
N3 (xN3, yN3, zN3) Bǰ%vQf C5N1 'vQf C5N3 BȠÔ (Fig.13A)ǈ

ȵvQf Pn +ȠÔ*,?< 2 #+vQfBǰŐ (5) +;*İȸ(Fig.13B)A1T1G2G3C3 Ěė??+ǈȵvQf Pn +Ù*#%,n &ȍPn (xn, yn, zn) = C5N1 × C5N3= (xN1 – xC5, yN1 – yC5, zN1 – zC5) × (xN3 – xC5, yN3 – yC5, zN3 – zC5)
= ((yN1 – yC5)•(zN3 – zC5)  (zN1 – zC5)•(yN3 – yC5),(5)(zN1 – zC5)•(xN3 – xC5) – (xN1 –xC5)•(zN3 – zC5),(xN1 – xC5)•(yN3 – yC5)  (yN1 – yC5)•(xN3 – xC5))

Fig.13 (A) Vector C5N1 and vector C5N3 in A, T, G, or C. (B) Normal vector Pn was calculated
from C5N1 and vector C5N3 (Equation (5)). Reproduced from Suzuki M. et al., Molecules, 19,11030-11044 (2014) with permission from MDPI.
29

ƺ*G2 ' A1 (θ (G2–A1))G2 ' T1 (θ (G2–T1))A1 ' T1 (θ (A1–T1))G2 ' G3 (θ (G2–
G3))G2 ' C3 (θ (G2–C3))G3 ' C3 (θ (G3–C3)) ʪ+ɨ 6 #+ʽɤB (Fig.14)?
?+Ěė+ǈȵvQfBǰ%ɨȠ (Ő (6)-(11))

Fig.14 Calculated dihedral angle θ (G2–A1), θ (G2–T1) and θ (A1–T1) showed red arrows, and thecalculated dihedral angle θ (G2–G3), θ (G2–C3) and θ (G3–C3) showed blue arrows. Reproduced
from Suzuki M. et al., Molecules, 19, 11030-11044 (2014) with permission from MDPI.

θ (G2–A1) = arccos (PG2 • PA1 / |PG2| |PA1|)(6)

θ (G2–T1) = arccos (PG2 • PT1 / |PG2| |PT1|)(7)θ (A1–T1) = arccos (PA1 • PT1 / |PA1| |PT1|)(8)

θ (G2–G3) = arccos (PG2 • PG3 / |PG2| |PG3|)(9)

θ (G2–C3) = arccos (PG2 • PC3 / |PG2| |PC3|)(10)θ (G3–C3) = arccos (PG3 • PC3 / |PG3| |PC3|)

(11)

30

DNA ʣʧ+ƽ6+ƀƷ'%ƽ6Ŏù

δ1 'δ3 BŐ (12) 'Ő (13) +; *İȸδ1 = θ (G2–A1) + θ (G2–T1) + θ (A1–T1)(12)

δ3 = θ (G2–G3) + θ (G2–C3) + θ (G3–C3)

(13)

31

Ȝ 3 ȡ ȱƭ'ȺĹ
3-1. G:X ĚėĺB 1 #ü7 DNA ƨʧ+Ƶʓƣʛç
Ȝ 2 Ț*%REiʢçƈ¿ (Oz 5, IaSp) 'REiĚėĺ+ĭİçJkPBɨȠIa ' Sp , sp3 ǝȫBƤ%>8S ­' R ­>+ɨȠȱƭ,“G:X (X = Oz 5, S-IaR-IaS-SpR-Sp)” Ěėĺ+ĭİŪ G:S-Ia > G:R-Ia>> G:R-Sp > G:S-Sp >> G:Oz &>'BȜ 2 Ț*ȍƨȚ&,DNA z]+ǍŪţÍ*>G:X ĚėĺBü7 DNA ƨʧ+ĭİŪ+ʙ>'Bɬ6hPol β +ȱƟƵʓ (PDB entry: 1BPY) 40) Bǰ

Bƛ<

~
*

%G:X ĚėĺB?

? 1 #ü7 hPol β-DNA eB®ų5G:X Ěėĺ+Ƶʓ,Ȝ 2 Ț+ɨȠȱƭ*%ƣ9ĭİ&"ĚėĺƵʓBǰʤĨçĪɨȠ,ÕĨÞĪɨȠ;=9İʤŪ*Â?%
G:X ĚėĺʟÕ,Ȝ 2 Ț&ŝƣʛçƵʓBǰ*Ġ+ïĨBüC&>ƨȚ&,ƣ9ʣɝ&>hPol β-DNA e,ʼŋ>8ʤĨçĪɨȠ*;>Ƶʓƣʛç,ǫıǼ)Ɲʪ&ɨȠ

&hPol β-DNA e+Ƶʓƣʛç*,ÕĨÞĪɨȠBǰ&)#5=

ƣ9ʣɝ) G:X ĚėĺʟÕ+Ƶʓ*+6ʤĨçĪɨȠBhPol β-DNA eÉ­+Ƶʓƣʛç*,ÕĨÞĪɨȠBǰƨȚ&,G:X Ěėĺ'+ 5'

Ěėĺ*ȅȀ (Fig.12Fig.15)Fig.12 *ȍ;;/ 3'½+ʴƄ*ţ+ G:X ĚėĺB“G2X2”

X + 5'½+ A:T ĚėĺB“A1T1” X + 3'½+ĚėĺB“G3C3”'¥ʮ,ȍ5ŝ<? A1T1G2X2G3C3 B Fig.15 *ȍ+ 3 #+Ěėĺ

<)> DNA ƨʧ+JkPBȠÔ>'&X2 +ŖʿBǂʊ>'Bɬ6Ɛ??ǵ)"%X2 ,ïĨƐ+>+&X2 ĚėBü7Ƶʓ+JkP,ȁƄǂʊ&)&??+Ƶʓ+&Ëʑ>ʟÕ&> A1T1 ' G3C3 *ȅȀ+ G2X2BʰËʑʟÕ+JkPBȠÔǂʊ>'&ʢçƈ¿Bü7'&ǯ> DNA ƨʧ+ĭİçBɫ³

32

Fig.15 Minimized geometries of “A1T1, G2X2, G3C3” containing X2 = (A) C, (B) Oz, (C) S-Ia,(D) R-Ia, (E) S-Sp, or (F) R-Sp as viewed from the minor groove. Reproduced from Suzuki M. etal., Molecules, 19, 11030-11044 (2014) with permission from MDPI.
33

3-2. X + 5'½;/ 3'½+Ěėĺ+ĭİçJkPƈ¿Ěė*;> DNA ƨʧ+ĭİçBɫ³>8*??+ƈ¿Bü7
DNA ƨʧ+“G2X2”ĚėĺBʰËʑ>ʟÕBǰ%ĭİçJkPBȠÔ

3-2-1. X

5'!& $%

X2 + 5'½Ěėĺ&>“A1T1”Ěėĺ (ΔE1) +ĭİçJkP,Ő (2) *;"%
İȸȠÔ (Table 3)5Ȅȗ+ĭİçJkP,ΔE1DFTǄ+ĭİçJkP,ΔE1SCRF &ȍΔE1DFT 'ΔE1SCRF ,'9* R-Sp > S-Sp > S-Ia > Oz >R-Ia &"
Kornyushyna <,ʒŎɶɥƫãǪɍ'BĘþ%<Gh/Ia *ĺ>REi+õ=ʎ6, Sp ;=9> 26)+ɨȠȱƭ,ƈ¿+ 5'½Ěėĺ&>“A1T1”,ș­ǵŪ­*ʫ´) Ia +ƕ Sp ;=9ĭİ&=Kornyushyna <+Ęþ'Ɋ%)<Oz *#%, R-Ia ;=9ΔE1 ºġĭİ&"#

Table 3 Destabilization energies (kcal/mol) of “A1T1” (ΔE1), “G3C3” (ΔE3), and “A1T1 + G3C3”(ΔE1 + 3), each value was calculated with minimized geometries. Reproduced from Suzuki M. etal., Molecules, 19, 11030-11044 (2014) with permission from MDPI.

Xa

A1T1

G3C3

A1T1 + G3C3

Δ E1DFT Δ E1SCRF

Δ E3DFT DE3SCRF

Δ E1+3DFT Δ E1+3SCRF

Oz

1.3

1.1

−0.1

0.6

1.1

1.0

S-Ia

1.4

1.3

2.6

1.3

4.1

4.5

R-Ia

0.5

0.6

4.0

3.9

4.8

4.6

S-Sp

2.1

2.1

2.7

2.8

4.8

5.3

R-Sp

12.6

12.4

5.3

4.5

18.3

18.3

a

X = the damage contained in the minimized structure.

34

5=5'½Ěėĺ&>“A1T1”+ĭİŪ&,Ia ;=9 Oz +ƈ¿=ʄų+ƕãǪǼ&"¥Ü+ıˋȱƭ 20) Bɳƛ>',&)DNA ù"&

ƺ* 3'½Ěėĺ&>“G3C3”+ĭİçJkPBɨȠ>'*

3-2-2. X

3'!& $%

X2 + 3'½Ěėĺ&>“G3C3”Ěėĺ (ȄȗːΔE3DFTǄːΔE3SCRF) +ĭİçJkP,Ő (3) *;"%İȸ?ʤĨçĪɨȠ*;"%ȠÔ (Table 3)+ȱƭĭİçJkPΔE3DFT;/ΔE3SCRF ,'9* R-Sp > R-Ia > S-Sp > S-Ia > Oz')"ˌ2'*X2 + 3'½Ěėĺ&>“G3C3”Ěėĺ+ĭİçJkP, Oz ƣ9ľ º&>'<“G3C3”Ěėĺ, Oz ƣ9ĭİ&>'ȍ?
X2 + 3'½Ěėĺ&>“G3C3”Ěėĺ+ĭİŪ,Ia : Sp ;=9 Oz +ƈ¿=ʄ
DNA ùų+ƕãǪǼ&"¥Ü+ıˋȱƭ 20, 26) 'Ɋ%



ɨȠȱ

ƭ, S-Sp +“G3C3”9 R-Ia +“G3C3”;=9ĭİ&>'Bȍ% =X2 + 3'½Ěė
ĺ&>“G3C3”Ěėĺ+ĭİŪBǰ
"Ŝ"%ƺ* X2 + 5'½%ʘð+ıˋȱƭ 20, 26) BĮÉ*,ɳƛ&);/ 3'½ĚėĺBü7ƵʓBǰ%ĭİçJkPBȠÔ

3-2-3. G2X2 ; X

5'

3'

!& ,925$%


G2X2 ĚėĺBʰËʑʟÕ&>“A1T1”;/“G3C3”ĚėĺBü7“A1T1 + G3C3”(ȄȗːΔE1+3DFTǄːΔE1+3SCRF) +ĭİçJkPBɨȠ?<,Ő (4)&İȸ (Table 3)ΔE1+3DFT
 Ia , Sp ;=9ΔE1+3DFT;/ΔE1+3SCRF +ºƣ9ľ

+, Oz &"5;/ΔE1+3SCRF +ºľș­ǵŪ­*ȅȀ>'Ia' Sp *Ëʑ% S ­+ƕ R ­;=9ľ "¥B5'8>'ΔE1+3DFT , R-Sp
> S-Sp~R-Ia > S-Ia > OzΔE1+3SCRF , R-Sp > S-Sp > R-Ia > S-Ia > Oz &>Oz +“A1T1 + G3C3”,¢+ƈ¿+“A1T1 + G3C3”;=9ĭİ&"?,ĚėĺBü
7 DNA Ƶʓ¢+ƈ¿ĚėĺBü7 DNA Ƶʓ*ǂ2ĢǠ+ĚėĺBü7 DNA Ƶ35ʓ*ˈªŪˏ'Bȍ%>Ƚƞ&9ʐ2ĢǠ+f^QcQĚėĺ*;=Ƶų?> DNA ƨʧ;=9ʼŌʽ+ DNA ƈ¿Bü7 DNA 3(ƨʧƵʓƽC&ĭİç 39)DNA z<?%~],ƽC

DNA Bɜɛ*'Ȉ> 37)#5=&ɨȠȱƭ,Ia : Sp ;=9 Oz +ƕƈ¿=ʄ

ùų:

'

DNA

ıˋȱƭ 20, 26) Bɳƛ&>9+&"

R-Ia ' S-Sp +ΔE1+3DFT º*#%,ıˋȱƭ 26) 'Ɋ%

"



ıʳ+ DNA ɜɛ*ʫ>ȊȖ,Ǆǘǐ&ɕA?>8 20, 26)DNA ɜɛ*

>DNA ƨʧ+ĭİŪ,Ǆ*>'Ⱥ)

>ĭİçJkP&>ΔE1+3SCRF º*ʂČ<?>ıʳ*?<+ΔE1+3SCRF ºB6>'R-Ia +ƕ S-Sp ;=9¬ĭİ&>'Bȍ%=¥Ü+ıˋȱƭ 26) 'Ɋ><*S-Sp*ʫ%,in vivo *% S-Sp +ƕ R-Sp ;=9mGnZãǪɍ';/ R-SpĘþ= 41)ƨɨȠȱƭ,?Bɳƛ&>9+&"¥B5'8>'X2 + 5'½Ěėĺ&>“A1T1”Ěėĺ5, X2 + 3'½Ěėĺ&>“G3C3”Ěėĺ

&, G:Oz 5, G:IaG:Sp ĚėĺB 1 #ü7 DNA ƨʧ+ĭİŪBɫ³>*,èÕ&=+ƕ+ĚėĺBü7Ƶʓ&>“A1T1 + G3C3”Bǰ%ţ* G:X ĚėĺBü7??+>'Ťɝ&>'Ľƺ˂*DNA ƨʧ*Ěėĺ*

%Oz 5, S-IaR-IaS-SpR-Sp “A1T1”Ěėĺ>Ŗʿ*#%Ƴɩ>8DNA +ƽ6+n~`çBɕ"

3-3. G:C ĚėĺBü7 DNA ƨʧ
Fig.15<+ƽ6Ŏù

< Oz 5, S-IaR-IaS-SpR-Sp B 1 #ü7 DNA ƨʧ+ƽ6Ŏù

ŉ>;
ƽ6Ŏù

;/“G3C3”)ɠɡǼ)íɺBö+ʙŻɺǼ)ɠɡǼíɺ&,øƵʓʪ+Bɮȭ*ǂʊ>*,èÕ&>8?<+ɠɡǼíɺBÌ­

Ǽ*n~`ç>'*Ő (12) :Ő (13) +;3-3-1. 5'!&

*

. (

36

)Ő&İȸ>''G2X2 Ěėĺ+ 5'½Ěėĺ+ƽ6ŎùBǇİ>8G2 ' A1 (θ (G2–A1))G2 ' T1

(θ (G2–T1))A1 ' T1 (θ (A1–T1)) ʪ+ɨ 3 #+ʽɤBŐ (6)-(8) Bǰ (Fig.14 +ʀȇí)%ƽ6Ŏù

%??ɨȠ(δ1) BŐ (12) *ȍ;*θ (G2–A1)'θ (G2–T1)θ (A1–T1) +ùɨº'%İȸȠÔd1 , S-Sp > R-Sp > S-Ia > Oz >C > R-Ia ')" (Table 4)

δ1 +º < R-Ia *ʴƄ>“A1T1”Ěėĺ+ƽ6,ƣ9ľ+Ƶʓ,ĢǠ+Ěėĺ<Ƶų?>Ƶʓ'ˈª%ĭİçJkPΔE1 ƣ9¬

 (Table 4;/ Fig.15A15D)Ŝ"%R-Ia +<?><*ΔE1 +ġS-Ia > Oz

"'Ⱥ

> R-Ia ,ƽ6Ŏù δ1 'Ɋ% #5=δ1 ġ '“A1T1”Ěėĺˇɑ*ƽ
C&>'Bȍ%'>=?, X2 Ěė*;"%őʂ?>'Bȍ%R-Sp ' S-Sp +ΔE1 º*#?>ƽ6Ŏù

%%,??+ƈ¿*;"%őʂδ1 &,ɳƛ&) "<*Ia ' Sp +ƕ*Ëʑ%S ­+ΔE1 º, R ­+ΔE1 º;=9ġ "S-Ia
+ƕû*û

>

;/ S-Sp + C1'~bė“A1T1”Ěėĺ

= (Fig.15C15E)X2 + 5'½*%, S ­+Ŗʿ, R ­;=9ġ"¥ <ƽ6Ŏù δ1 &,¥Ü+ıˋȱƭ 20, 26)9ĮÉ*,ɳƛ&)&)ΔE1 º*#%"Table 4 Dihedral angles θ (G2–A1), θ (G2–T1), and θ (A1–T1) (red arrows in Fig.14), and thedegree of distortion δ1. Reproduced from Suzuki M. et al., Molecules, 19, 11030-11044 (2014)
with permission from MDPI.
Xa

θ (G2–A1)

θ (G2–T1)

θ (A1–T1)

C

25.2°

12.5°

13.3°

51.0°

Oz

19.4°

18.2°

18.0°

55.6°

S-Ia

38.3°

28.2°

11.3°

77.8°

R-Ia

3.3°

4.0°

4.0°

11.3°

S-Sp

56.1°

29.9°

26.4°

112.4°

37

δ1

R-Sp
a

10.9°

32.4°

43.2°

86.5°

X = the damage contained in the minimized structure.

38

3-3-2. 3'!&. (X2 Ěė+ 5'½Ěėĺ+ƽ6Ŏù

(3-3-1) 'úƶ*G2 ' G3 (θ (G2–G3))G2 ' C3 (θ(G2–C3))G3 ' C3 (θ (G3–C3)) ʪ+ɨ 3 #+ʽɤBŐ (9)-(11) Bǰ%??ɨȠ (Fig.14 +ʻȇí)5ƽ6Ŏù*θ (G2–G3)(δ3) BŐ (13) *ȍ;'θ (G2–C3)θ (G3–C3) +ùɨº'%İȸȠÔδ3 , R-Ia > R-Sp > S-Sp > S-Ia
> C > Oz ')" (Table 5)
X2 Ěė+ 3'½Ěėĺ+ƽ6Ŏù δ3 ,δ1 ',ǵ)=Oz *ʴƄ>“G3C3”Ěėĺƣ9ƽ6ľĢǠ+Ƶʓ'ƣ9ˈª%<*ΔE3 +ġS-Sp > S-Ia > Oz ,ƽ6Ŏù (Table 5;/ Fig.15A15B)

δ3 +˃'Ɋ% 3.3.1 +δ1 'úƶ*G3C3 Ěė+ĭİç, G2X2 ĚėïČ')"%ǯ>ƽ6*;"%ɳƛ>%,Ia ' Sp '9* R ­, S ­;=9'&ƕδ1 ',ǵ)=δ3 º*ġR-Ia;/ R-Sp + C1'~bė“G3C3”Ěėĺ+ƕû*û15F);"%R-Ia;/ R-Sp + C3  R-Ia%

 (Fig.15D

;/ R-Sp + C1'~bė*;"%Ǧ*ƽ

8<?'ȍ?ƽ6Ŏù δ3 ,δ1 'úƶ*¥Ü+ıˋȱƭ 20, 26)ºBĮÉ*ɳƛ>',&)

;/ΔE3

"

Table 5 Dihedral angles θ (G2–G3), θ (G2–C3), and θ (G3–C3) (blue arrows in Fig.14), and thedegree of distortion δ3. Reproduced from Suzuki M. et al., Molecules, 19, 11030-11044 (2014)
with permission from MDPI.
Xa

θ (G2–G3)

θ (G2–C3)

θ (G3–C3)

C

9.5°

8.0°

17.2°

34.6°

Oz

11.1°

3.1°

11.9°

26.1°

S-Ia

5.4°

13.9°

19.2°

38.5°

R-Ia

17.9°

42.6°

49.2°

109.7°

S-Sp

5.5°

25.7°

23.2°

54.4°

R-Sp

22.9°

36.2°

43.3°

102.5°aδ3

X = the damage contained in the minimized structure.
39

3-3-3. *!

5'

ƈ¿Ěė X2 + 5'½

3'!&+7)64.

(

;/ 3'½ĚėĺƕBȺűδ1 'δ3 +ùɨº'%ȴùǼ)ƽ

(“δ1 + δ3”) B&Ɣ*İȸ“δ1 + δ3”, Table 6 *ȍ;

6Ŏù*R-Sp> S-Sp > R-Ia > S-Ia > C > Oz ')=??+ƈ¿*ʫ%,ΔE1+3 'Ɋ%“δ1 + δ3”*ȴùǼ)ƽ6Ŏù
;=9ƽ6Ŏù

%,Oz , C ;=9ºľOz ,ĢǠ+ DNA

'ȍ?ΔE1+3

Ŀ)



<“G2Oz2”*ʴƄ>“A1T1 + G3C3”

Ěėĺ,ĢǠ+“G2C2”*ʴƄ>“A1T1 + G3C3”Ěėĺ;=9ĭİ&>'ȍ?%=Ȇȃ%

#5=+ȴùǼ)ƽ6Ŏù+ĭİŪBʟÕǼ*ɳƛ&)“δ1 + δ3”&

“A1T1 + G3C3”"Table 6 Total degree of distortion (“δ1 + δ3”). Reproduced from Suzuki M. et al., Molecules, 19,11030-11044 (2014) with permission from MDPI.
Xaa

δ1 + δ3

C

85.6°

Oz

81.7°

S-Ia

116.3°

R-Ia

121.1°

S-Sp

166.8°

R-Sp

189.0°

X = the damage contained in the minimized structure.

3-3-4. :*"1C

 . ( '/8

3-3-3 &ʐ2“G2Oz2”'“G2C2”ʪ* >ƽ6Ŏù

“δ1 + δ3”'“A1T1 + G3C3”+ĭİŪ+ɊBɥǇ>'Bɬ6ëȩ*ƽ6ŎùBȍ“δ1 + δ3”Bǂʊ>¤A=*ĭİçJkP+ȠÔ'úƶ* C +“δ1 + δ3”ºBėǗ'+ėǗ'?
40

?+ƈ¿'+ŉBȠÔ+ȱƭC ' S-Ia 5, R-Ia +“δ1 + δ3”º+ŉ, 30.7°
35.4°&"?*ĺ%C ' Oz +“δ1 + δ3”º,A

4.0°+ŉ)"#5=“G2Oz2”*ʴƄ>“A1T1 + G3C3”+ƽ6,“G2S-Ia2”5,“G2R-Ia2” *ʴƄ>“A1T1 + G3C3”;=9ľ'Bȍ%=Fig.15 *>ɠɡǼ)íɺ'9Ɋ<*C ' S-Sp 5, R-Sp +“δ1 + δ3”º+ŉ, 81.1°103.3°&=“G2S-Sp2”%

5,“G2R-Sp2” *ʴƄ>“A1T1 + G3C3”ėǗ'“G2C2” <ƣ9ƽC&

>'

Bȍ%

?*#%9Fig.15 +ɠɡǼ)íɺ'Ɋ%+ėǗ'

“G2C2”<+ƽ6Ŏù

*ʫ>ȺĹ,“G2X2”*ʴƄ>“A1T1 + G3C3”+ĭİŪBɳƛ>'&Üʐ“δ1 + δ3”º*ß
6Ŏù

δ1 º

;/δ3 º*ʫ%9 C BėǗ'ėǗ<+ƽ%Ƴɩ+ȱƭδ1 º*ʫ%, S-Sp >'%??+ƈ¿*#R-Ia > R-Sp > S-Ia > Ozδ3 º&, R-Ia > R-Sp > S-Sp > Oz > S-Ia ')"#5=δ1 º;/δ3 º*ʫ%9 C BėǗ*ǂʊɠɡǼíɺ'Ɋ“δ1 + δ3”º',ǵ)"%δ1 º;/δ3 º,ɠɡǼíɺ',Ɋ)¥B5'8>'δ1 º;/δ3 º&,ʟÕǼ*º,δ1 º'δ3 ºBùɨ>'&
]Bǰ

"

+ŒǞBɚ

ɳƛ&)

ù"%

"“δ1 + δ3”=¥Ü+ DNA z~ıˋȱƭ 20, 26) 'ĭİçJkPΔE1+3 Bɳƛ>'&ɨȠ

ȱƭ*;>'“G2X2”Ěėĺ*ʴƄ>“A1T1 + G3C3”+Ƶʓƈ¿+ʼŌʽŪ*;>Ŗ
ʿ*;"%ƽC

'Ⱥ

<?>ɧ

Ƈ

>'REiʢçƈ¿Bü7 DNA ƨʧ+ĭİŪ,??+REiʢçƈ¿+ʼŌʽŪ*;"%Ǉ5>'

>

@ #

5=G:Oz ĚėĺBü7 DNA ƨʧ+ƽ6¢+ƈ¿Bü7 DNA ƨʧ;=9ľ',ùǭǼ&>'Ⱥ

<?>

41

Ȝ 4 ȡ ȱɶ
DNA dtf Oz +'+ƕ Ia : Sp +';=9REi+õ=ʎ6ãǪɍ

'Bʘð+ıˋȱƭ 20, 26) ȍ%;"%ȠÔ G:Oz
ãǪ+ʙ>Ȝ 2 Ț*

>ʤĨçĪɨȠ*;/ G:IaG:Sp Ěėĺ+ĭİŪ&,+REi+õ=ʎ6

Bɳƛ&)"&ƨȚ&,?<+Ěėĺ+ŌʽŪ+ʙȅȀ?<+ĚėĺBü7 DNA ƨʧ+ƽ6Ŏù

*+ŉ*;"%REi+õ=ʎ6ãǪBɳƛ>'Bɬ6ƨȚ&, 3 Ěėĺ<)=ţ*ĚėĺB“G2X2”ü7 DNA ƨʧeBǰ%

“G2X2”Ěėĺ¥ğ+ËʑʟÕBǰ%JkP+ǂʊBɕ"+ȱƭX2 +5'½+“A1T1”Ěėĺ+ĭİŪ*#%, R-Ia > Oz > S-Ia > S-Sp > R-Sp &=X2 + 3'½+“G3C3”Ěėĺ+ĭİŪ*#%, Oz > S-Ia > S-Sp > R-Ia > R-Sp ')"?<+ɨȠȱƭ&,ʘð+ıˋȱƭ 20, 26) Bɳƛ>',&)"&“A1T1” ;/“G3C3”ĚėĺBü7Ƶʓ“A1T1 + G3C3”+ĭİçJkPBȠÔ+ȱƭ
“A1T1 + G3C3”+ĭİŪ, Oz > S-Ia > R-Ia > S-Sp > R-Sp ')"#5=X2 Ěė+ 5'½' 3'½+ƕ*ʴƄ>ĚėĺBȺű>'&ʘð+ DNA z~]Bǰ

ıˋȱƭ 20, 26) Bɳƛ>'&<*??+ƈ¿*;>ŖʿBƳɩ>8DNA ƨʧ+ƽ6Ŏù~`çX2 Ěė+ 5'½+ƽ6Ŏù δ1 ' 3'½+ƽ6Ŏù

Bn

δ3 ,“A1T1”'“G3C3”

Ěėĺ+ĭİŪ ;/ʘð+ıˋȱƭ 20, 26) Bɳƛ&) " X2 Ěė+ 5'½' 3'½ƕ+ƽ6Ŏù+ùɨº&>“δ1 + δ3”&,ĢǠ+ G2C2 ĚėĺBü7 DNA'ǂʊ>'&“δ1 + δ3”º, R-Sp >> S-Sp >> R-Ia > S-Ia >> Oz ')"+“δ1 + δ3”º,“A1T1 + G3C3”+ĭİŪ'Ɋ%¥B5'8>'ʼŌʽ+ƈ¿ĚėB

ü7 DNA ,ƽ6:ĭİç:%ȱƭǼ*ʼŌʽ+ƈ¿&,Ōʽ)ƈ¿;=9 DNA z<G:Oz~]+mGnZãǪ¬>'Ⱥ<?>ƨȚ+ȱƭ;/ G:IaG:Sp Ěėĺ*ʴƄ>Ěėĺ*;"%Ƶų?ƵʓBǰ

>',?<+ĚėĺBü7 DNA ƨʧ+ĭİŪBɫ³>+*èÕ&>'BȍG:Oz ü7 DNA ƨʧƵʓ+ƽ6, G:Ia : G:Sp Bü7 DNA +ƽ6;=942

"?ƎG:Oz , G:Ia : G:Sp ;=9 DNA zľDNA ùų+ãǪɍ"'Ⱥ~]*;>ƈ¿=ʄ<?>ƨȚ&,ÕĨÞĪɨȠ*;>Ƶʓƣʛç*,Ěėĺ:`nQɾ
+eLOX

yZ

;/

ʢJZd9ü7eBǰ<+e,ʼŋ*Ġ+ïĨBüC&dBü5)zeBǰ)<?

=ǫıǼ)ɨȠƝʪ&,ʤĨçĪɨȠBɕ8ʤĨçĪɨȠ*, DNA +eLOX

'&)

&)DNA

yZ

;/

ʢJZ

Ŝ"%?<+ DNA ƈ¿*;"%ǯ>ƽ6 DNA~]*;> DNA ùų*>Ƽȋ)ŖʿBɳƛ>8*,ĚėĺÍ+ƽ6ȥ+ș­Ƶʓ*(+ȒŎ+ĞçBőʂ+
ˆ&>

43

BȺű>'

Ś+Ƴɩɴ

Ȝ4Ț
Oz *ĺ>Ěė+õ=ʎ6ɥƫ(ƨȚ+ÍĶ*ʫ%, Chem. Res. Toxicol., 28, 1307-1316 (2015) *ǻɘ6)
Reprinted (adapted) with permission from “Suzuki M., Kino K., Kawada T., Morikawa M., Kobayashi T.,
and Miyazawa H. (2015) Analysis of nucleotide insertion opposite 2,2,4-triamino-5(2H)-oxazolone byeukaryotic B- and Y-family DNA polymerases. Chem. Res. Toxicol., 28, 1307-1316. (DOI:
10.1021/acs.chemrestox.5b00114)”. Copyright 2015 American Chemical Society.

Ȝ 1 ȡ Ƚƞ
Ȝ 1 Ț&9ʐ2?5& DNA z~]óŦɥƫȱƭ, G:C-C:G fZmY+ǻǯƸƵɥƛ*ƈ¿'% Oz *ȅȀ%>Ȝ 3 Ț+ȱƭƨʧ DNA +Ƶʓ'ˈª%< 20, 26)ƨȊȖ&

>ʣɝŎ Gh/Ia : Sp ;=9ˏ
<9Oz Bü7ƈ¿ DNA ,ĢǠ+>'ǖ?Oz *ĺ>REi+õ=ʎ6*;= G:C-C:G fZmYǻǯ>'ȍĄ?+8G:C-C:G f
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DNA ɜɛ*ʫ> DNA zȓˈĩđ>DNA z
DNA ɜɛ, Pol α~],ġɄɐ&, 5 ȓˈof*~]*;"%+)Ƹȿ,ǵ)"%;/ Pol γPol δPol ε *;"%ɕA?%

Pol δPol ε + 3 ȓˈ+ DNA z~],ƮɎ­*ż"%> (Table 7) 42-44)5DNA z

ȂúŪ

<

D|

#

*ł%

+rD|

=Pol α> 42, 43)Ǧ*Pol α~]ǍŪ+ǍŪţ+;/ Pol δPol ε , B r> 42, 43)Pol α-tG{]ɜù­,tG{+ùų'©ʨ*ʫ 42)Pol δ ,PRʧ+ùųB 45)Pol ε ,> 46)5Pol γ , A rD|
DNA z>Ȅưǯǥ+> DNA ɜɛ+ţǼŗÝB

~],z*Õˈ?%

"%, 15

*ł%

eFRʧ+ùųB*ż"%=|fUg

E DNA +ɜɛBɕ~]&> (Table 7) 43)ɌǼ*ƶ)ɝČ*;"%ǯ>ƈ¿ DNA, DNA z~]*;> DNA ùųBʭĴDNA ɜɛƸƵB¼ƻ%544&DNA ɜɛ+¼ƻBċʞ>8*ȭȾ,ƈ¿B=ʄ
> DNA zǥ*~]B¾

Pol ζ , B-rD|ˈ?%;/ Pol ηPol ιPol κREV1 ,Ȅưǯ% >Pol ζ

DNA ùųBż

%ƈ¿=ʄ

* 42, 43, 48)Pol ηDNA z~]&> (Table 7) 43, 47, 48);/ Pol ιPol κREV1 , Y rD|> (Table 7) 42, 43, 48, 49)Oz Bü7ƈ¿ DNA +ɜɛƸƵBƛ<,5,ƣ×+ȊȖɴˆ'%DNA ɜɛBżDNA ùųBż
Ŧɥƫ% DNA Bùų&

DNA z

DNA z

~]+ƕ+ DNA z*Õ*>8*~]'ƈ¿=ʄ~]*;>ƈ¿=ʄó;/Ěėõ=ʎ6ɥƫʣɝ')>Ȝ 1 Ț&9ʐ2;ʎ6ɥƫ*Pol α;/ƈ¿=ʄ;/ Pol γPol εPol η + Oz *ĺ>Ěė+õ=óŦ,Ɨ*ɥƫ?%> 20, 21)Pol α;/ Pol ε , Oz

*ĺ%REiBPol γ ,REi'EeiBPol η ,REi'EeiX
fXBõ=ʎ6?<É%+ DNA z~] Oz B=ʄ% DNA Bùų>'&>ƨȚ&,Oz *ʫ%Ʀɥƫ&" Pol δPol ζPol ιPol κREV1+ Oz *ĺ> DNA z~]óŦBɕOz *ĺ>ƈ¿=ʄóŦ

ėõ=ʎ6óŦBɥƫ

Table 7 The function of DNA polymerases 42, 43).
function

DNA polymerase

familyPol γ

A-family

Pol αReplicative DNA polymerases

Pol δ

B-family

Pol ε
Pol ζ
DNA polymerases

Pol η

involved in translesion

Pol ι

synthesis

Pol κ
REV1
45

Y-family;/ĚȜ 2 ȡ ıˋƕǈ2-1. L

VjQLbgʘð+Ɠǩ 20) BñȺ*DNA z~]óŦ*ǰ  Oz Bü7 30-mer +L

V

jQLbg (5'-CTCATCAACATCTTXAATTCACAATCAATA-3'X = Oz) Bɵɛ(Fig.16)ƕGh Bü7 30-mer +L VjQLbg (X = Gh) ,ćȨÇÑƏƃ;=Ɔ±Bö 22)DNA z~]óŦ+Uf'%ǰ

(Fig.17) Bü7 30-mer +L

REi5, 8-oxoGTHF

VjQLbg (X = G 5, 8-oxoGTHF)

țB Alexa680 &Ʒɹç?L;/ 5'Ƨ

VjQLbg(5'-*TATTGATTGTGAATT-3') ,Ƙƨ

mGLWpZ (Saitama, Japan) ;=ɿÈ

Fig.16 Outline of the preparation of 30-mer DNA containing the Oz lesion.

O

O

H

O

Fig.17 Structure of THF.

46

2-2. DNA z~]yeast Pol ζ (yPol ζ);/ yeast REV1 (yREV1), Enzymax (Lexington, KY)

<ɿÈ


Human Pol (hPol) δ BƵų> 4 #+Wsicf (p125p50p66p12) ,?
? POLD1POLD2POLD3POLD4 *;"%Ug?%>p125 +Uphf`nQB®ɛ>8* (Fig.18)C Ƨț* 6×His `Rɔù p125 BUg
 >  ġ Ʉ ɐ ǻ ǫ ǰ v Q `  pTriEx-1.1/p125-His6 B Ƶ Ȣ  
pTriEx-1.1/p125-His6 Bȯ6ʎC(Fig.19A) ġɄɐ Rosetta2 (DE3) pLysS * 1 mM IPTG Bß%ǻǫɰĽǻǫɰĽġɄɐ, 25 ºC & 3 ƝʪĖˉŚʚţÕʷ*;=ċô-80ºC *ÒȱµĩÒȱġɄɐB lysis mcrD (20 mM sodium phosphate (pH 7.4),
0.5 M NaCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) *ÎŲǛʃʾǉÓǭŚʚţÕʷ*;"%ȭȾǘɥǐBŝŝ<?ȭȾǘɥǐB HisTrap HP
1 ml (GE Healthcare, Tokyo, Japan) *ÄěP40 mcrD (20 mM sodium phosphate(pH 7.4), 0.5 M NaCl, 40 mM imidazole, 0.5 mM PMSF) &ǌǏŚP500 mcrD (20mM sodium phosphate (pH 7.4), 0.5 M NaCl, 500 mM imidazole, 0.5 mM PMSF) BǰÔp125 Bü7ǘÔǐ, vivaspin 15 (Sartorius, Tokyo, Japan) Bǰ%ǘʯğ@ʘ*;= hPol δ µĩǰmcrD (25 mM Bis-Tris-HCl (pH 6.5), 0.5 mM EDTA, 0.1 mM EGTA,
20% glycerol, 1 mM DTT, 0.5 mM PMSF) *ȷƇĿʤ#ÕǊ%-80 ºC *µĩ
hPol δ ɜù­+Uphf`nQ,ʘð+Ɠǩ 50) BñȺ*®ɛ (Fig.18

Fig.19)5p125 BUg>ġɄɐǻǫǰvQ`pTriEx-1.1/p125N Ƨț* 16×His' FLAG `Rɔù p12pCOLADuet-1/p50-p66-p12;/ p50p66 BUg>ġɄɐǻǫǰvQ`B Ƶ Ȣ   (Fig.19B)  pTriEx-1.1/p125

pCOLADuet-1/p50-p66-p12 + 2 #+ġɄɐǻǫǰvQ`Bȯ6ʎC(DE3) pLysS * 1 mM IPTG Bß; /ġɄɐ Rosetta2%ǻǫɰĽǻǫɰĽġɄɐ, 16 ºC & 12 ƝʪĖˉŚʚţÕʷ*;=ċô-80 ºC *Òȱµĩ¥ʮ+Ɗ®, p125 'úƶ*ɕ

Ŀʤ#ÕǊ%-80 ºC *µĩ

47

Fig.18 Construction of recombinant hPol δ. (A) p125 (without the p50, p66 and p12 subunits),and (B) hPol δ full complex.

Fig.19 Expression and purification (A) of p125 (without the p50, p66 and p12 subunits), (B) of
hPol δ full complex. hPol δ subunits (p125, p50, p66, and p12) are encoded by cDNAs POLD1,
POLD2, POLD3, and POLD4, respectively.

48

hPol ι , POLI *;"%Ug?%=+ÕĨʤ, 83 kDa &>N Ƨț*

GST `Rɔù hPol ι BUg>ġɄɐǻǫǰvQ`pGEX6P1/hPol ι BƵȢ (Fig.20)pGEX6P1/hPol ι Bȯ6ʎC
BßġɄɐ Rosetta2 (DE3) pLysS * 1 mM IPTG

%ǻǫɰĽǻǫɰĽġɄɐ, 25 ºC & 3 ƝʪĖˉŚʚţÕʷ*;=ċô-80 ºC *ÒȱµĩÒȱġɄɐB binding mcrD (PBS (pH 7.3); 140mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.5 mM PMSF) *ÎŲǛʃʾǉÓǭŚʚţÕʷ*;"%ȭȾǘɥǐBŝŝ<?ȭȾǘɥǐB GSTrap HP 1 ml
(GE Healthcare) *Äěbinding mcrD&ǌǏŚPG mcrD (50 mM Tris-HCl(pH 7.4), 10 mM reduced glutathione, 0.5 mM PMSF) Bǰǐ, vivaspin 15 (Sartorius) Bǰ%ǘÔhPol ι Bü7ǘÔʯğ@ʘ*;= hPol ι µĩǰmcrD (50 mM

Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol (2-ME), 0.5 mM
PMSF) *ȷƇĿʤ#ÕǊ%-80 ºC *µĩ

Fig.20 Expression and purification of hPol ι.
49hPol κ , POLK *;"%Ug?%=+ÕĨʤ, 99 kDa &>N Ƨț*

6×His `Rɔù hPol κ BUg>ġɄɐǻǫǰvQ`pET15b/hPol κ BƵȢ (Fig.21)pET15b/hPol κ Bȯ6ʎC ġɄɐ Rosetta2 (DE3) pLysS * 1 mM IPTG Bß%ǻǫɰĽǻǫɰĽġɄɐ, 25 ºC & 3 ƝʪĖˉŚʚţÕʷ*;=ċ

ô-80 ºC *ÒȱµĩÒȱġɄɐB lysis mcrD*ÎŲǛʃʾǉÓǭŚʚţÕʷ*;"%ȭȾǘɥǐBŝŝ<?ȭȾǘɥǐB HisTrap HP 1 ml (GEHealthcare) *ÄěP40-T mcrD (20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 40
mM imidazole, 0.01% Triton X-100, 0.5 mM PMSF) &ǌǏŚ<* P100-T mcrD
(20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 100 mM imidazole, 0.01% Triton X-100, 0.5
mM PMSF) &ǌǏ+Ś P200-T mcrD (20 mM sodium phosphate (pH 7.4), 0.5
M NaCl, 200 mM imidazole, 0.01% Triton X-100, 0.5 mM PMSF) Bǰ
Bü7ǘÔǐ, vivaspin 15 (Sartorius) Bǰ%ǘÔhPol κʯğ@ʘ*;= hPol κ µĩǰmcrD

 (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50% glycerol, 1 mM DTT, 0.5 mM PMSF) *ȷƇ-20 ºC *µĩ

Fig.21 Expression and purification of hPol κ.
50Human REV1 (hREV1) , REV1 *;"%Ug?%
>REV1 ,ÕĨʤġ8ġɄɐǻǫȦBǰ

phfB®ɛ>*,ǻǫʤ¬%Éʨ

<)> REV1 +U'ǖ?>&ãǪɍ REV1 +Uphf+®ɛ*,ʘð+Ɠǩ 51) BñȺ*341

ǍŪB9#
ʢ

=+ÕĨʤ, 138 kDa &< 829 +E|l<Ƶų?>ƹģĞǵ­ hREV1(341-829)B®ɛ (Fig.22)ƹģĞǵ­ hREV1(341-829)+ÕĨʤ, 65 kDa &>N Ƨț* 6×His `Rɔù hREV1(341-829)BUg>ġɄɐǻǫǰvQ`pET15b/ hREV1(341-829)BƵȢ (Fig.23)pET15b/ hREV1(341-829)Bȯ6ʎCġɄɐ Rosetta2 (DE3) pLysS * 1 mM IPTG Bß%ǻǫɰĽǻǫɰĽġɄɐ, 25 ºC & 3 ƝʪĖˉŚʚţÕʷ*;=ċô-80 ºC *Òȱµĩ¥ʮ+Ɗ®, Pol δ 'úƶ*ɕhREV1(341-829)µĩǰmcrD (50 mM HEPES-NaOH(pH 7.5), 0.5 M NaCl, 10% glycerol, 10 mM 2-ME, 0.5 mM PMSF) *ȷƇĿʤ#ÕǊ
%-80 ºC *µĩ

Fig.22 Structure of hREV1 and the region used for construction of recombinant of
hREV1(341-829).

51

Fig. 23 Expression and purification of REV1(341-829).

52

2-3. CBB ƮɎ®ɛøUphf*ǘɥmcrD (62.5 mM Tris-HCl (pH 6.8), 10% glycerol,

2% SDS, 0.05 mg/ml bromophenol blue) Bß5 Õʪ 95 ºC &ÓǭSDS-PAGE &ÕʷŚTB CBB Stain One (nacalai tesque, Kyoto, Japan) &ƮɎLAS3000 (Fujifilm,
Tokyo, Japan) Bǰ%ɥƫ

2-4. HIZ`scdFRǈ
CBB ƮɎ'úƶ*®ɛø Uphf*ǘɥmcrD (62.5 mM Tris-HCl(pH 6.8), 10% glycerol, 2% SDS, 0.05 mg/ml bromophenol blue) Bß5 Õʪ 95 ºC &ÓǭSDS-PAGE &ÕʷŚfZrDmcrD (25 mM Tris, 189 mM glycine,
10% methanol) & Immobilon-P transfer membrane (Merck Millipore, Darmstadt, Germany)* Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA) &ʈÏ5%ZO}|Q&scORŚ1 ƺŷ­BĲǕ& 1 ƝʪóŦ0.1% PBS-T (137 mM NaCl,
2.7 mM KCl, 8.1 mM Na2HPO4•12H2O, 1.47 mM KH2PO4, 0.1% Tween 20) &ǌǏŚ2 ƺŷ­ B Ĳ Ǖ & 1 Ɲ ʪ  ó Ŧ     0.1% PBS-T & ǌ Ǐ Ś  Immobilon Westernchemiluminescent HRP substrate (Merck Millipore) & Ƴ Ô    ɥ ƫ * , LAS3000(Fujifilm) Bǰ1 ƺŷ­'%Anti-His-tag (MBL, Nagoya, Japan)Anti-DNA

polymerase δ p125 catalytic subunit (MBL)Anti-DNA polymerase δ p50 small subunit (MBL)
ŷ DNA  δ  p66 ŷ ­ (Bio Academia, Osaka, Japan) 
ANTI-FLAG® M2 antibody (Sigma-Aldrich, St. Louis, MO), Anti-GST (Mouse IgG2a-κ),
Monoclonal (GS019), AS, POD Conjugated (nacalai tesque) B  2 ƺ ŷ ­ '  %Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch,West Grove, PA), Goat F(ab')2 Fragment Anti-Rat IgG (H+L)-Peroxidase (BECKMANCOULTER, Brea, CA) B¯ǰ53

2-3. DNA z~]óŦ

DNA z~]óŦ*ǰóŦǐ+ȯųBƺ*ȍp125;/ hPol δ ɜù­+óŦǐ+ȯų, 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 2 mM DTT, 100 µg/mL bovine
serum albumin (BSA)ˑ yPol ζ;/ hPol κhREV1(341-829)yREV1 +óŦǐ+ȯų,

50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 5 mM DTT, 100 µg/mL BSAˑhPol ι +óŦǐ+ȯų, 40 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 150 mM NaCl, 1 mM DTT, 10 µg/mL BSA, 10%
glycerol &>hPol δ ɜù­;/ yPol ζhPol ιhPol κhREV1(341-829)yREV1 +óŦǐ, 100 fmol + DNA dtf;/ 50 fmol + 5'ƧțB Alexa680 &Ʒɹç

tG{Bp125 +óŦǐ, 100 fmol + DNA dtf;/ 50 fmol + 5'ƧțB[32P]&ƷɹçtG{BüC&;/ DNA z>??+ dNTP]+ǜŎ,??+ď+ɳƛƓ*ɪʋtG{©ʨóŦ~;/Ěė+õ= DNA dtf'tG{;/ dNTP +ȯ6ùA, Fig.24*ȍ¥Bü7óŦǐ,ÉʤB 5 µl 'p125;/ hPol δ ɜù­yPol ζyREV1

ʎ6ɥƫ*ǰ

+óŦǐ, 30 ºC & 30 ÕʪhPol ι +óŦǐ, 30 ºC & 15 ÕʪhPol κ +óŦǐ, 37 ºC& 30 ÕʪóŦBɕ

5 µl + stop mcrD (15 mM EDTA, 10% glycerol) BßŦB¼ƻ%8 M ŀȫBü7 16%z

EQ

Tris, 89 mM boric acid, 2 mM EDTA) mcrDBǰ+! 2.5 µl B 30 W & 90 ÕʪʹǃǋäBɕ%ó

E|gT' 1×TBE (89 mM%??+óŦŚ+WtÕʷʹǃǋäŚÕʷT+! 5'ƧțB Alexa680 &ƷɹçtG{Bü7Wt, Odyssey® Infrared
Imaging System (LI-COR, Lincoln, NE) B5'ƧțB[32P]&ƷɹçtG{Bü7W
t*#%, BAS2500 bioimaging analyzer (Fujifilm) B¯ǰ%ɥƫ

54

Fig.24 Experimental design of DNA polymerase assay. The 15-mer primer is labelled with
Alexa 680 or [32P] indicated by ‘*’.

2-4. ʒŎɶɥƫʘð+Ɠǩ 16) BñȺ*hREV1(341-829)+ G+õ=ʎ6ãǪBɥƫDNA z
4-20%+ãǪ')>;;/ OzTHF8-oxoGGh *ĺ> dCTP~];/ dCTP ,dCTP +õ=ʎ6*ǜŎBɵƑʒŎɶɥƫ**, 2-3 'úƶ+óŦǐ+ȯųBǰ

> DNA z~]óŦ

30 ºC & 1 Õʪ5, 2 ÕʪóŦBɕ"óŦ

Ś+Wt, 2-3 'úƶ*ɥƫKm º' Vmax º, DNA z~]óŦB 2 ċ

5, 3 ċɕ"ȱƭ%ȠÔ

<GHFmmQtcfBǰ55

Ȝ 3 ȡ ȱƭ'ȺĹ
3-1. hPol δ + Oz *ĺ> DNA z*Pol δ , Pol α : Pol ε 'ú B rD|

Üʐ;

DNA z

ţǼŗÝBżsicf~]óŦɥƫ~]&>hPol δ , p125p50p66p12 + 4 #+W<)>ɜù­&=ɦħWsicf, p125 &> 52)ƨȊȖ&,

ɦħWsicfë­&> p125
ǰ%Oz B=ʄ

20);/ 4 #+Wsicf% DNA Bùų&>

Oz *ĺ% dGTP Bõ=ʎ7;/ 4 #+WsicfQ*ł> DNA ɜɛ+((<)> hPol δ ɜù­B

5Pol α : Pol ε 'úƶ*BɥƫɦħWsicfë­&> p125<)> hPol δ ɜù­, UphfB®ɛz

E|gTʹǃǋäŚ+ CBB ƮɎ;/HIZ`scdFRǈ*%ȋɯ (Fig.25 ' Fig.26)®ɛ hPol δ ɜù­+ȩŎ,¬ŝ<?ʤ9Ŀ)
8øWsicf+ĩđǂ*#E%,Øİ&)""

Fig.25 Expression and purification of recombinant p125. (A) p125 was electrophoresed andstained with CBB. (B) p125 was identified by immunoblotting with anti-His antibody.

56

Fig.26 Expression and purification of recombinant hPol δ full complex. (A) hPol δ full complex
was electrophoresed and stained with CBB. (B) hPol δ full complex was identified byimmunoblotting with anti-p125, anti-p50, anti-p66, anti-FLAG antibody.

5ɦħWsicf&> p125 Bǰ
Oz B=ʄ%ɥƫ'@ʼŋ*¬ãǪ)<%Éʨ+ 30-mer 5& DNA B©ʨ%Ěė+õ=ʎ6ɥƫȱƭ (Fig.27A + 5)5

<p125 ë­&, G *ĺ% dCTP BƼȋ*õ=ʎ7+*ĺ

% (Fig.27B + 1)Oz *ĺ%, dGTP Bõ=ʎ7'ƛ<(Fig.27B + 7)<*hPol δ ɜù­*#
B=ʄ

*)"

%9 p125 ë­+ęù'úƶ*Oz%Éʨ+ 30-mer 5& DNA B©ʨ (Fig.28A + 5)Oz *ĺ%9dGTP BÂ«*õ=ʎC&

(Fig.28B + 7)#5=hPol δ ,úrD|

+ Pol α : Pol ε 'úƶ* 20)Oz *ĺ% dGTP Bõ=ʎ7'ƛ<

57

')"

Fig.27 DNA synthesis with p125 across Oz. (A) Primer extension across Oz by p125.
Decreasing amounts of p125 (96.5 ng in lanes 1 and 5, 9.65 ng in lanes 2 and 6, 0.965 ng in lanes
3 and 7) were incubated with template (containing G in lanes 1-4 and Oz in lanes 5-7) in thepresence of each of the four dNTPs (500 µM each). (B) Nucleotide selectivity of p125 opposite
Oz. p125 (9.65 ng) was incubated with template (containing G in lanes 1-5, and Oz in lanes 6-9)in the presence of 500 µM of a single dNTP (N = C, G, A or T) (lanes 1-4 and lanes 6-9).
Copyright 2015 American Chemical Society.

58

Fig. 28 DNA synthesis with hPol δ full complex across Oz. (A) Primer extension across Oz by
hPol δ full complex. Decreasing amounts of hPol δ full complex (154 ng in lanes 1 and 5, 15.4
ng in lanes 2 and 6, 1.54 ng in lanes 3 and 7) were incubated with template (containing G inlanes 1-4 and Oz in lanes 5-7) and each of the four dNTPs (100 µM each). (B) Nucleotide
selectivity of hPol δ full complex opposite Oz. hPol δ full complex (154 ng) was incubated with
template (containing G in lanes 1-5, containing Oz in lanes 6-9) and 100 µM of a single dNTP
(N = C, G, A or T) (lanes 1-4 and lanes 6-9). Lane 4 in panel A and lane 5 in panel B containedno enzyme and are negative controls. Copyright 2015 American Chemical Society.

59ɦħWsicf&> p125 , 3'-5'JO^jQE]ǍŪBƤ%δ ,ŧıŎ+ˏDNA z~]'%Ȉ<?%>8hPol>+JO^jQE]Ǎ

Ū, DNA ɜɛ+ƯƼ:ʛƼĚėĺ+·ƼŃŅrR~f+ųǣç)(*ʫ> 53)ƨɥƫȱƭ*%%9G Bü7 DNA dtf+ęù'ǂʊ>' Oz

Bü7 DNA dtf+ęù&,3'-5'JO^jQE]ǍŪ*;"%Õɥ?
tG{+ʤĜß% (Fig.28A + 5 ' Fig.28A + 1 BFig.28B+ 6-9 ' Fig.28B + 1-4 Bǂʊ)#5=hPol δ + 3'-5'JO^jQE]ǍŪB9"%%9Oz Bü7 DNA +ɜɛ*',&)

>ɲõ=ʎ6BĮÉ*ƯƼ>

'ȍ?Ƚƞ&9ʐ2;*Oz ,REi+ʢçƈ¿&>'<Oz *ĺ% dGTPÂ«*õ=ʎ5?>'*;=G:C-C:G fZmYőʂ?>5
?5&+ Oz *ĺ>Ěė+õ=ʎ6ɥƫ*ǰ
dGTP +õ=ʎ6Ëʑ% >'*ß

20, 21)<? DNA z~]É%*

Ȝ 2 Ț&ȍ;*ʤĨçĪɨȠ<9 Oz ' G ĭİ)ĚėĺBŕų>'Bǖ%=hPol δ  Oz *ĺ%dGTP Bõ=ʎ7',ŭʑ=&"#5=hPol δ , Oz Bü7 DNA +ɜɛ*%G:C-C:G fZmYBɰǻ>'ȍĄ?

60

3-2. yPol ζ + Oz *ĺ> DNA z~]óŦɥƫPol ζ ,ɲ=!)ƈ¿=ʄ

DNA ùų*ʫ%*z%ʣɝ)ŗÝBż"%~]&> 47, 54, 55)

> DNA z~]ǍŪ,ƈ¿*;"%ǵ)>'G{ (6-4) ǯųǥB=ʄ+ãǪ,ʼŋ*¬

ǦŢ>°56)% DNA ùųBɕi (O6-methylguanineːO6-MeG) *#=UV *;>ȘǠĞǵɰǻ-Pol ζ ,b|aƕ8-oxoG : O6-~bRE% DNA ùųBɕ> 57)5THF :XQs`ēq|Y%,ƈ¿ĚėB=ʄ

'Ęþ?%+ DNAaG{ (cyclobutane pyrimidine dimerːCPD) (Fig.29) &,Ȃɚʧ½*ĚėB3'C(õ=ʎ5ƈ¿ʟ«& DNA ùų¼ƻ%5&yPol ζ  Oz B=ʄ% DNA ùųBɕ

O

(

*#

%ɥƫO

HNO' "Ęþ9> 56, 58)

NH
N

N

dR

dR

O

Fig.29 Structure of CPD.

+ȱƭyPol ζ , Oz B=ʄ%Éʨ5&ãǪɍùų (Fig.30A + 5-7)

+ãǪ, G Bü7 DNA dtf+ęù'+ŉ,A 5-7 ' 1-3 Bǂʊ)¥Ü+Ęþ*&" (Fig.30A +>¢+ DNA z~] 20, 21) :Ü˂+ hPol δ +ęù&,Oz B=ʄ

 DNA ùųãǪ, G B=ʄ*ǂ2>'¬?<+ DNA z~]*ǂ2>' yPol ζ +ȱƭ,ˌ2ȱƭ&"#5=¢+ DNA z~]',ǵ)=Oz , Pol ζ *;> DNA ùųãǪ*3'C(ŖʿB) DNA ùųãǪ'ȍĄ?

ƺ*yPol ζ  Oz *ĺ%õ=ʎ7ĚėBɥƫUf'%ǰ

G

Bü7 DNA dtf+ęùG *ĺ% dCTP ƣ9Â«*õ=ʎ5? (Fig.30B
61

Fig.30 DNA synthesis with yPol ζ across Oz. (A) Primer extension across Oz by yPol ζ.
Decreasing amounts of yPol ζ (50 ng in lanes 1 and 5, 17 ng in lanes 2 and 6, 5.0 ng in lanes 3
and 7) were incubated with template (containing G in lanes 1-4, containing Oz in lanes 5-8) andeach of the four dNTPs (100 µM). Lanes 4 and 8 contained no enzyme as the negative control.
(B and C) Nucleotide selectivity of yPol ζ opposite Oz. yPol ζ (17 ng) was incubated with
template (containing G in B, and Oz in C) and 100 µM of a single dNTP (N = C, G, A or T)(lanes 1-4). Lane 5 contained no enzyme as the negative control. (D and E) Primer extension andnucleotide selectivity of yPol ζ opposite Oz. yPol ζ (1.7 ng) was incubated with template(containing G in D, and Oz in E) and 100 µM of each of the four dNTPs (lane 2) or 100 µM of asingle dNTP (N = C, G, A or T) (lanes 3-6). Lane 1 contained no enzyme as the negative control.
Copyright 2015 American Chemical Society.
62+ 1) dGTP ' dTTP +õ=ʎ696<? (Fig.30B + 2 ' 4)dATP +%,18-mer 5, 20-mer 5&©ʨmgƳÔ? (Fig.30B

õ=ʎ6*#+ 3)?*ĺ%Oz Bü7 DNA dtf+ęù,Oz *ĺ% dGTP
=Oz + 5'½ʴƄĚė&> T *ĺ%9 dGTP Ŀõƣ9Â«*õ=ʎ5?%

 (Fig.30C + 2)Oz *ĺ> dATP +õ=ʎ6, G Bü7 DNA=ʎ5?%dtf+ęù'úƶ* 18-mer 5, 20-mer 5&©ʨmgƳÔ?%=+ʤ,Ĝß% (Fig.30C + 3)ƕOz *ĺ> dCTP;/ dTTP+õ=ʎ6,ǔĿ (Fig.30C + 1 ' 4)ʡȫǜŎB¬ęù9úƶ+Àû6<? (Fig.30D ' 30E)?<+ȱƭ<Pol ζ , G%9ɲ=!)Ěėõ=ʎ6Bɕ

')"

'ƛ<

&) Oz *ĺ*Pol ζ , THF *ĺ%3'C(ĚėBõ=ʎ5)Üʐ;: O6-MeG ,REi'Ƶʓª%
DNA ùųãǪ,ʼŋ*¬

'ƛ<

>*9

Pol ζ ¥ğ+ DNA z

')"%

'Ⱥ

<?%

>ıʳ*

% DNA BãǪǼ*ɜɛ>8*,<ƈ¿B=ʄ~]ƈ¿*ĺ%ĚėBõ=ʎ7Ťɝ=+Ś Pol ζ)<ƨȊȖȱƭ,ˌ2'*yPol ζ  Oz *ĺ%ĚėBõ=ʎC&ƈ¿B=ʄ

% DNA Bùų*+ãǪĢǠ+ G Bü7 DNA dtfBǰ34úȒŎ&>'Bȍ+'ğBõ=ʎ7ɲ=!) DNA zùų*> 57)#5=Pol ζ ,ƈ¿+ȓˈ DNA B©ʨ>'ȍĄ?% 47, 56, 59-61)

>'Bƛ<58-oxoG

A<?<+ƈ¿B=ʄ*ʫ´)ƈ¿Ěė*ĺ>Ěė+õ=ʎ6,ʼãǪǼ?5&+ĠȊȖȱƭ

58)

ęù'

Oz *ĺ% dCTP ¥<yPol ζ '~]&>'%9Oz +ƈ¿=ʄ

% yPol ζ ʣɝ)ŗÝBż"%

63

>+

9?)



DNA

3-3. hPol ι + Oz *ĺ> DNA z~]óŦɥƫPol ι ,ɲ=!) DNA ùųBɦħ> DNA z+ȊȖ~]+ 1 #&>?5&<in vitro &+Ěėõ=ʎ6ɥƫ&,ɲõ=ʎ6ɟ<?% =°-G*

ĺ> dTTP +õ=ʎ6&> 62-65)5Pol ι +ǦŢǼ)ǍŪ'%ŌĒ% 2 Ěė

< 3 ĚėȒŎtG{B©ʨ)

hPol ι (Fig.31)  DNA z~]ǍŪ

Bü7 DNA dtfBǰ=ʎCǞ<?> 66)&®ɛ;/?<+ǦŢBƤ%

>

(

G

%ȋɯ+ȱƭdCTP ' dTTP B G *ĺ%õ

' (Fig.32A + 3, 5)

;/tG{B 2 Ěė5, 3 Ěė©ʨmgȋɯ? (Fig.32A + 2)®ɛ hPol ι ,hPol ι *ǦŢǼ) DNAz~]ǍŪBƤ%

Fig.31 Expression and purification of recombinant hPol ι. (A) hPol ι was electrophoresed andstained with CBB. (B) hPol ι was identified by immunoblotting with anti-GST antibody.

64

Fig.32 DNA synthesis with hPol ι across Oz. hPol ι (0.5 µg) was incubated with template(containing G in A, and Oz in B) and 100 µM of each of the four dNTPs (lane 2) or 100 µM of asingle dNTP (N = C, G, A or T) (lanes 3-6). Lane 1 contained no enzyme as the negative control.
Copyright 2015 American Chemical Society.

ƺ*Oz *ĺ> Pol ι +Ěė+õ=ʎ6ɥƫBɕ"+ȱƭOz *ĺ> dGTP;/ dATP +õ=ʎ6 G Bü7 DNA dtfBǰęù'ǂ2>'Ĝß (Fig.32B + 4, 5)dCTP +õ=ʎ6,¬ (Fig.32B + 3)ƕ
dTTP +õ=ʎ6, G Bü7 DNA dtfBǰęù'34úȒŎ&=(Fig.32B + 6)?, dTTP +õ=ʎ6 hPol ι ǦƤ+ǍŪ&=DNA dtf+ȓˈ*²ĩ)'BŮĀ%5¥Ü+ȊȖ&,hPol ι 'úY-family *ł> hPol η  dGTP ' dATP Bõ=ʎC&= 21)hPol ι  hPol η +n

R&>'9Ⱥű>' 67)hPol ι  hPol η 'úƶ* dGTP ' dATP Bõ=ʎC
'Ⱥ

<?>

hPol ι , Oz *ĺ%ĚėBõ=ʎC
 2)?5&+ȊȖ+Ś+©ʨóŦ,¼ƻ (Fig.32B +<hPol ι , 8-oxoG *ĺ%ĚėBõ=ʎCŚ8-oxoG+ʴƄĚė*ĺ%9ĚėBõ=ʎ7'&>ƕTHF +ęù, THF +ʴƄĚ
ė*,ĚėBõ=ʎ7'&)'Ęþ?%> 68)hPol ι + Oz *ĺ>DNA ©ʨóŦ' THF *ĺ> DNA ©ʨóŦ+Àû,ª%% DNA Bùų&)Ū, yPol ζ ;=9¬''Ⱥ>hPol ι , Oz B=ʄ<Oz Bü7ƈ¿ DNA +ɜɛ*

<?>
65

> hPol ι +ʣɝ3-4. hPol κ + Oz *ĺ> DNA z~]óŦɥƫPol κ *, 2 #+ǦŢǼ)ǍŪ>'?5&+ȊȖ&ƛ<
1 #8,Pol κ , DNA dtf+Ƨț

*)"%

>

< 1 #5, 2 #ŴÜ& DNA ùų¼

ƻ>Ǟ&> 69-71)+ïČ,tG{' DNA dtf+|ZEG~f&>' Wolfle <*;"%Ęþ?%*ĺ>Ěė+õ=ʎ6*> 72)2 #8,Ěėõ=ʎ6Ǧ*REi%Pol κ ,ƼĚė'ɲ"Ěė+ƕBõ=ʎ7

Ǟ&> 69-71)9!@CREi*ĺ> dCTP +õ=ʎ6, dGTP : dATPdTTP*ǂ2>';=ãǪǼ&>'ʒŎɶɥƫ
hPol κ (Fig.33) +ȩŎ,ˏ)

<ƛ<

')"%

> 70)®ɛ?<+ 2 #+ǦŢǼ)ǍŪBƤ%G Bü7 DNA dtfBǰ%ȋɯ+ȱƭ®ɛ hPol κ Éʨ 30-mer+ DNA dtf+ 1 ĚėŴÜ&>, 29-mer 5&ãǪɍ©ʨ%+ 1)5G *ĺ% dCTP Bõ=ʎ7+*ß
: dATPdTTP 9õ=ʎC&+ǦŢǼ)ǍŪBƤ%>'B

 (Fig.34A(Fig.34A + 2)dGTP= (Fig.34A + 3-5)?5&+Ęþ*> 2 #>'ȋɯ?ƕOz Bü7 DNA dtfBǰęù,29-mer 5&©ʨ%

(Fig.34B + 1)+ãǪ, G Bü7 DNA dtf*ǂ2%ʼŋ*¬"(Fig.34B + 1 ' Fig.34A + 1 Bǂʊ)5Oz *ĺ%, dGTP Bƣ9;õ=ʎ6 (Fig.34B + 3)ƺ* dATP õ=ʎ5?:
 4)dCTP : dTTP +õ=ʎ6,ƳÔ?)" (Fig.34B +" (Fig.34B + 2 ' 5)

?<+ȱƭ,Pol ι 'úƶ* Pol κ ,??ë­&, Oz Bü7 DNA +ƈ¿=DNA ùų*ʫ)ʄbR*;=ƈ¿=ʄ

'Ⱥ

<?>

¢+ DNA z~]'+ZGc

DNA ùų*ʫ>÷ȿŪ9>8?<*# %,ŚȺű>Ťɝ>

66

Fig.33 Expression and purification of recombinant hPol κ. (A) hPol κ was electrophoresed andstained with CBB. (B) hPol κ was identified by immunoblotting with anti-His antibody.

Fig.34 DNA synthesis with hPol κ across Oz. hPol κ (9.2 ng in A and B) was incubated with
template (containing G in A, and Oz in B) in the presence of 100 µM of each of the four dNTPs(lane 1) or 100 µM of a single dNTP (N = C, G, A or T) (lanes 2-5). Lane 6 contained no
enzyme as the negative control. Copyright 2015 American Chemical Society.

67

3-5. hREV1(341-829);/ yREV1 + Oz *ĺ> DNA z

REV1 , Y rD|*ł%> DNA zfZrI]'%9Ȉ<?%
ZrI]*;=G
EOREi'~]óŦɥƫ~]&=eLOXXbYʢ> 49, 73)REV1 ,+eLOXXbYʢf&) A : CT<*,ɀĚėƈ¿: 8-oxoGN2-"ƶ)ƈ¿*ĺ%9 dCTP Bõ=ʎ7'ƛ<')"> 73-76)5hREV1 + R357  dCTP 'Ǆȫȱù>'&dCTP Bõ=ʎ7Ǧ%ŢǼ)~Mi[}ȱƟƵʓɥƫ*;=ƛ<*)"%')> G 'õ=ʎ5?> dCTP ȁƄǄȫȱù)> 77)#5=dtf+&REV1 ,dtf+Ěė*²ĩ dCTP Bõ=ʎ7'&>+&>
ƨȊȖ&,ʘð+ƓǩBñȺ*eLOXXbYʢfZrI]ǍŪBȍ341< 829 +E|lʢ<Ƶų?>ƹģĞǵ­ hREV1(341-829)B®ɛ (Fig.35)DNAz~]óŦɥƫ*ǰ

Fig.35 Expression and purification of recombinant hREV1(341-829). (A) hREV1(341-829) was
electrophoresed and stained with CBB. (B) hREV1(341-829) was identified by immunoblottingwith anti-His antibody.

68

5®ɛ hREV1(341-829) G;/ THF8-oxoG *ĺ>Ěė+õ=ʎ6ɥƫ*;=?<+ƈ¿*ĺ% dCTP BÂ«*õ=ʎ7eLOXXbYʢfZrI>'Bȋɯ (Fig.36B ' 36D36E + 3)ƺ*Oz]ǍŪB9"%*ĺ>Ěė+õ=ʎ6Bɥƫȱƭ?5&*ɥƫ?¢+ DNA z~]',ǵ)=hREV1(341-829), Oz *ĺ% dCTP BÂ«*õ=ʎ7'Bƛ<*(Fig.36C + 3)<*Oz 'úƶ* G:C-C:G fZmY+ïČ')>REiʢçƈ¿+ 1 #&> Gh/Ia *ĺ%9hREV1(341-829), dCTP BÂ«*õ=ʎ
(Fig.36F + 3)#5=hREV1 ,eLOXXbYʢfZrI]C

ǍŪ*;= Oz : Gh/Ia *ĺ% dCTP Bõ=ʎ7'&Oz : Gh/Ia<ǯ> G:C-C:GfZmYBċʞ&>'ȍĄ?
ƺ*dNTP +ǜŎBĜßęù+G
Ěė+õ=ʎ6ɥƫ;/ OzTHF8-oxoGGh/Ia *ĺ><REV1 *;>ƈ¿'+óŦŪ+ʙ+ƤǟBƛ<

'Bɬ6+ȱƭdNTP +ǜŎBĜßęùhREV1(341-829), G*>&) G+ 5'½ʴƄĚė*ĺ%9 dCTP Bõ=ʎ6 (Fig.37A + 3) dGTP : dTTP +ɲõ=ʎ69ɟ<? (Fig.37A + 4 ' 6)5hREV1(341-829), 8-oxoG + 5'½ʴ
ƄĚė*ĺ%9 dCTP Bõ=ʎC&

 (Fig.37D + 3)ƕOz : THFGh/Ia+ęù, G +ęù'úƶ*dGTP ' dTTP +ɲõ=ʎ69ɟ<? (Fig.37B;/

37C37E + 4 ' 6)ƈ¿Ěė+ 5'½ʴƄĚė*ĺ> dCTP +õ=ʎ6,6<?
" (Fig.37B);/ 37C37E + 3)#5=ƈ¿Ěė+ 5'½ʴƄĚė*ĺ

>Ěė+õ=ʎ6,ʭĴ?%=ƈ¿ʟ«&ĮÉ* DNA ùų¼ƻ>'ȍĄ?5?<+ȱƭ<hREV1(341-829)+ Oz ' THFGh/Ia *ĺ>Ěė

+õ=ʎ6+óŦŪ,úÀû&>'ƛ<
<*yREV1 Bǰ')"% hREV1 'úƶ* Oz *ĺ% dCTP BÂ«*õ=ʎ7

Bɥƫ'@yREV1 9 Oz *ĺ% dCTP BÂ«*õ=ʎ7'ƛ<(')" (Fig.38B + 3)5+ãǪ, THF *ĺ> dCTP +õ=ʎ6ãǪ;=9A

*ɍ" (Fig.38B + 3 ' 38C + 3 Bǂʊ)¥

,of

ʡǁ

*²ĩ*Oz *ĺ% dCTP BÂ«*õ=ʎ7'&Oz<ǯ> G:C-C:G fZmYBċʞ&>'ȍĄ?
69<REV1

Fig.36 DNA synthesis with hREV1(341-829) across Oz. (A) Interaction between incoming dCTP
and R357 of hREV1. The structure of the dCTP:R357 pair was determined from the X-ray
crystal structure of hREV1 in a ternary complex with DNA and dCTP 77). (B-F) hREV1(341-829)(1.7 ng) was incubated with template (containing G in B, and Oz in C, THF in D, 8-oxoG in E,
Gh/Ia in F) and 1 µM in B, C and F, 10 µM in D and E of each of the four dNTPs (lane 2) or 1
µM in B, C and F, 10 µM in D and E of a single dNTP (N = C, G, A or T) (lanes 3-6). Lane 1
contained no enzyme as the negative control. Copyright 2015 American Chemical Society.

70Fig.37 DNA synthesis with hREV1(341-829) across lesions with 100 µM of dNTP. hREV1(341-829)(1.7 ng) was incubated with template (containing G in A, Oz in B, THF in C, 8-oxoG in D, Gh/Ia
in E) and 100 µM of each of the four dNTPs (lane 2) or 100 µM of a single dNTP (N = C, G, Aor T) (lanes 3-6). Lane 1 contained no enzyme as the negative control. Copyright 2015 American
Chemical Society.

Fig.38 DNA synthesis with yREV1 across lesions. yREV1 (12.5 ng) was incubated with
template (containing G in A, Oz in B, and THF in C) and 100 µM of each of the four dNTPs(lane 2) or 100 µM of a single dNTP (N = C, G, A or T) (lanes 3-6). Lane 1 contained no
enzyme as the negative control. Copyright 2015 American Chemical Society.

71

% DNA z

ƨȊȖ*;=?5&+ɥƫ*ǰ

~]+& REV1 ąOz *ĺ% dCTP BÂ«*õ=ʎ7 DNA z~]&>'ȍ?&

REV1 + Oz *ĺ> dCTP +õ=ʎ6'

ǦŢBƛ<

;= G

*>8*ʒŎɶɥƫ*;/ OzTHF8-oxoGGh/Ia *ĺ> dCTP +õ=ʎ6ãǪBƛ<*øƈ¿Ěė*ĺ> dCTP +õ=ʎ6ãǪ (fins) , G *ĺ> dCTP +õ=ʎ6ãǪ
(Vmax/ Km) '+ǂ'%ȠÔTHF;/ 8-oxoG + fins ,?? 0.030.01 &" (Table 8)#5=dCTP +õ=ʎ6ãǪ, DNA dtf*ü5?>Ěė G>> THF > 8-oxoG +˃*ɍ?,ʘð+Ęþ 51) 'Ɋ%?*ĺ%ˌ

2'* Oz + fins , 0.19 ' (Table 8)THF : 8-oxoG + fins *ǂ2>' 6 ¹9
, 19 ¹9ˏdCTP +õ=ʎ6ãǪ, DNA dtf*ü5?>Ěė Oz > THF> 8-oxoG +˃*ɍ'ƛ< ')"<*
Gh/Ia + fins , 0.12 &= (Table 8)

THF : 8-oxoG + fins *ǂ2>'ˏOz + fins ;=9¬"¥<dCTP +õ=ʎ6ãǪ, DNA dtf*ü5?>Ěė Oz > Gh/Ia > THF > 8-oxoG +˃*
ɍ

'ƛ<

')"

Table 8 Steady-state kinetics parameters for dCTP insertion opposite Oz, THF, 8-oxoG, Gh/Ia,
and unmodified control template G by hREV1(341-829). Copyright 2015 American Chemical
Society.
Km (µM)

Vmax/Km (s-1)

1.33 × 10-2

2.06 × 10-2

6.45 × 10-1

1Oz

1.15 × 10-2

9.48 × 10-2

1.21 × 10-1

0.19

THF

1.35 × 10-2

6.18 × 10-1

2.18 × 10-20.038-oxoG

1.21 × 10-2

1.70

0.71 × 10-2

0.01

Gh/Ia

1.62 × 10-2

2.07 × 10-1

7.82 × 10-2

0.12

template

Vmax (µM•s-1)

G

72

finsa

%REV1 *;> dCTP +õ=ʎ6+ɮȭ)~Mi[}Bǭɥ>8*Oz *ȳĺ> dCTP +õ=ʎ6ãǪ Gh/Ia : THF8-oxoG ;=9ɍ"ǭǱ*#%ȺĹhREV1 ' yREV1 (!<9 DNA dtf+ G ;=97@Ɉʇ+E|lʢ&>EPi (hREV1 , R357yREV1 , R324) BÚǰ%dCTP BãǪɍõ=ʎ7'ȱƟƵʓɥƫ

<Ɨ*ƛ<

')"%

> 77, 78)DNA dtf+ G, REV1 +GX (hREV1 , L358yREV1 , L325) *;"% DNA ucQZźÔ?<*,DNA dtf+ G +rRZdFʽ+ N7

<

;/ O6

ïĨ hREV1 &, H774 ' G775yREV1 &, M658 ' G686 +ʧ??Ǆȫȱ
ù>'*;=REV1 +ǍŪţ*>ǶǄŪzScf*Đİ?%77, 78)5?5&*ɕA?ʒŎɶɥƫ

6ãǪ,ɥʷİƐ*œ²ĩ%> (Fig.39A)<G ' 8-oxoG *ĺ> dCTP +õ=ʎ=DNA dtf G +ęù*ĺ> dCTP

+ȱùÞ*ǂ2>' DNA dtf 8-oxoG +ęù, dCTP +ȱùÞƤŮ*¬
>'ƛ<')"%> 51, 79)Howell <,¥ÜDNA dtf+ 8-oxoG+ H7 ' hREV1 + H774 +ʧǄȫȱù&) 'Bǖ%º,ɥʷİƐ&>+&Km ºġ?- DNA zŪ¬'Bȍ%> (Fig.39B) 79)Km~]&> REV1 '+ɣĂ>Table 8 *ȍʒŎɶɥƫȱƭB6>'Km º, 8-oxoG >Gh/Ia > Oz &=dCTP +õ=ʎ6ãǪ, Oz > Gh/Ia > 8-oxoG &"#5=Oz' hREV1 '+Ȃ®ǰ, 8-oxoG *ǂ2>'œ'<Oz , hREV1 +E|lʢ'ƨ+ǄȫȱùBŕų&>'ǖ&Oz +ƵʓB6%6>'Oz ,úŌʽ* G + N6;/ O6 ïĨ+¤A=')=> 2 #+udïĨB9"%>'<Oz +?<+ 2 #+udïĨ G +ęù'úƶ* hREV1 + H774 ' G775 'Ǆȫȱù&>'ǖ (Fig.39C)<*Gh ' hREV1 +Ȃ®ǰƶŐ, Gh '8-oxoG +Ƶʓ+ˈªŪ<8-oxoG ' hREV1 +Ȃ®ǰƶŐ'ª%>'ǖ&> (Fig.39D)+8Gh *ĺ> dCTP +õ=ʎ6ãǪ, 8-oxoG *ĺ> dCTP
+õ=ʎ6ãǪ'úȒŎ')>,&>ıʳ*, Table 8 *ȍ;*8-oxoG*ĺ> dCTP +õ=ʎ6*ǂ2>' Gh *ĺ> dCTP +õ=ʎ6,ãǪɍȜ 1 Ț&9ʐ2Gh , Ia 'ŌɗǧŰ*= (Scheme 1)É%+ pH *Ia ,ǒùǧŰ&ĩđ>'Ęþ?%73> 25)Ia , Oz +;* hREV1"% Gh '

A

R
G775
O

NH
H 2N

C
N
H
NH
N
HN

O
H

N
R
G775
O

O

REV1

H774

O
dR N
HN

N
O

X

H

R

N

H
H

NH

REV1

E
H 2N
dR N
HN

R

N
H

O
O

?

Gh

H774
O

N

H 2N

Oz

H774

D

R
G775
O

NH 2

X

NH

N

H

H

H 2N

O

O
O

N

H

8-oxoG

R

R

N

N

REV1

G

dR

dR N

N

H

O

N

O

H774
O

H

N

dR N

B

R

N

R
G775
O

REV1

H774
O

H

N

N

R
G775
O

H 2N

Ia

REV1

Fig.39 Proposed interactions between template bases and the main-chain amides of H774 andG775 of hREV1. (A) The N7 and O6 atoms of template G (Hoogsteen edge) interact with the
H774 and G775 main-chain amides of hREV1 77). (B) The interaction described by Howell etal.79) between an 8-oxoG and G775 of hREV1. Our proposed interaction between main-chain
amides of hREV1 and Oz (C), Gh (D) and Ia (E). The red X shows that there is no hydrogenbond. Copyright 2015 American Chemical Society.

74

'Ȃ®ǰ÷ȿ) 2 #+MyiǝȫBƤ%=?*;= Ia , hREV1 'ƨ+ǄȫȱùBŕųŝ>'Ⱥ <?> (Fig.39E) Ia , Oz ',ǵ)= sp3 ǝȫ
BƤ%

>8
Ia + O7 ïĨ,úŌʽ*)+ Ia + O7 ïĨ' hREV1 + G775

ʪ+Ǆȫȱù, Oz ' hREV1 +Ǆȫȱù*ǂ2>'Œ'ǖ?>"%Gh/Ia*ĺ> dCTP +õ=ʎ6 8-oxoG *ĺ> dCTP +õ=ʎ6;=9ãǪɍOz*ĺ> dCTP +õ=ʎ6ãǪ;=9¬"+,hREV1  Gh/Ia *ĺ% dCTP B

õ=ʎ7ʳ*Gh/Ia +Ōɗ, Ia ½*»"%

>+&,)

'Ⱥ

;/ Gh/Ia8-oxoG ' hREV1 '+Ȃ®ǰ*ʫ>ȺĹ+õ=ʎ6ʘȒ+ƈ¿Ěė+ǶǄŪzScf1+Đİ*%>¥+ Oz<hREV1 *;> dCTP%,Oz  Gh/Ia : 8-oxoG;=9ĭİ&>'ȍĄ?
ƨȊȖȱƭ<Oz *ĺ% REV1 Qf?>'>'REV1 REiʢçƈ¿&> Oz *ĺ% dCTP BÂ«*õ=ʎ7'&Oz<ǯ> G:C-C:G f

ZmYB,8'ǞȘǠĞǵBċʞ&>÷ȿŪBɟÔ?5&
Oz +·ş*ʫ>ȊȖ
Oz:G : Oz:A Ěėĺ<·şʡȫ&> NEIL1 : NTH1  Oz:C Ěėĺ<9 Oz Bõ=ʰ

%5

'ƛ<

')"%

8REV1 + Oz *ĺ> dCTP +õ=ʎ6,?<+ Oz:C ĚėĺƼȋ)·ş*%9ʣɝ')>'Ⱥ<+ Oz ʰð'REV1 , Oz *ĺ% dCTP
?5

<REV1 + C Ƨțg~GB¡%DNA +ɜɛBż

Pol δ :REV1 'úƶ*ƈ¿=ʄ

ē+ DNA z

Pol ιPol κ 'Ȃ®ǰ&>'ƛ<Oz 80)+

Ś+ DNA ùųóŦ,¼ƻ%5BÂ«*õ=ʎ7dCTP Bõ=ʎC&+ REV1 *ʫ>ȊȖĘþ

<?>

&)

')"%

~]&> Pol ζ : Pol η> 81-85) Ś+Ƴɩɴˆ'%

<ǯ> G:C-C:G fZmYBċʞ>~Mi[}Bƛ< *>8*REV1  Oz *ĺ% dCTP Bõ=ʎC]+ɥƫŤɝ&>

75

Ś+ DNA ©ʨóŦBż

DNA z

~

Ȝ 4 ȡ ȱɶ
Pol δ;/ Pol ιPol κ , Oz *ĺ%ɲ"ĚėBõ=ʎ6¬ãǪ&,> Oz

B=ʄ

% DNA Bùų (Table 9)Pol ζ , Oz *ĺ%ɲ=!)Ěėõ=ʎ6

Bɕ?5&ɥƫ?¢+ DNA z

34úȒŎ+ãǪ&Oz B=ʄ~]',ǵ)=ĢǠ+ G +ęù'

% DNA Bùų>'Bƛ<* (Table 9)

ƕRVE1 , Oz *ĺ% dCTP BÂ«*õ=ʎ7' < (Table 9)REiʢçƈ¿&> Oz<ǯ> G:C-C:G fZmYB,8'ǞȘǠĞǵBċʞ&>÷ȿŪBȑ8 DNA z~]&>'ƛ<ƭ,REV1 ' Pol ζ  Oz Bü7 DNA +ɜɛʘȒ*~]&>'ȍĄ?

%ʫ>ʣɝ) DNA z

Ś,?<+ DNA z+& Oz Bü7 DNA +ɜɛʘȒ*ʫ>ɥƫBʕ8>'Ťɝ&@')"¥ƨȊȖȱ(~]ıʳ*ȭȾ5+~Mi[}*#

Table 9 Summary of chapter 4 with previous results relating to Pols α, ε and η 20, 21).

Replicative DNA polymerases

DNA polymerases
involved in translesion synthesis

Insertion

Extension

Pol α

G 20)

low efficiency 20)

Pol δ

G 20)

low efficiency

Pol ε

G

low efficiency 20)

Pol ζ

G, A, C, T

high efficiency

Pol η

G, A, C 21)

low efficiency 21)

Pol ι

G, A, C, T

stall

Pol κ

G, A

low efficiency

REV1

C

stall

76

%

??+ DNA zȱƭ~] Oz *ĺ%õ=ʎ7Ěė*ȅȀ>'ƨȚ+ȊȖ;/ʘð+Ęþ 20, 21)<REV1 Bʰ DNA z

% dGTP Bõ=ʎ7'ƛ<+ˏɜɛē+ DNA z~]*#

Àû>'Ȉ<?%

õ=ʎ5?9+'Ⱥ

*ł%

>ŧıŎ

*Oz ' G +Ěėĺĭİ&>'*ʂČ>'Ⱥ
ē+ DNA z?>ƕƈ¿=ʄ

+ĭİŪ*#')"Ǧ*B rD|~] Oz *ĺ% dGTP +6Bõ=ʎ7',Ȝ 2 Ț;/Ȝ 3 Ț*ȍ;!) DNA ùųBɕ~]Ëʑ% Oz *ĺ<%,ŧıŎ¬ɲ==Oz *ĺ% dGTP ¥ğ+Ěė9<?>&ƺȚ&,Oz ' G 5, ATC +Ěėĺ%ɥƫ>''

77

Ȝ5Ț
Oz Bü7Ěėĺ+ĭİŪ+ DNA ©ʨóŦ*

>Ŗʿ(ƨȚ+ÍĶ*ʫ%, J. Nucleic Acids, 2014, 178350 (2015) *ǻɘ6)

Ȝ 1 ȡ ȽƞȜ 2 Ț&,Oz 5, Gh/IaSp 'REi+Ěėĺ+ĭİŪ*#Ȝ 3 Ț*%ʐ25%?<+ĚėĺBü7 DNA ƨʧ+ĭİŪ'ƽ6Ŏù

+ȱƭ*;"%DNA zBȠÔ~]*;>REi+õ=ʎ6ãǪ:ƈ¿=ʄ

DNA ùųãǪ Oz > Gh/Ia > Sp ')"¥Ü+ıˋȱƭ 20, 26) Bɳƛ>'&#5=Oz , DNA ƨʧ*

>ĭİŪ Gh/Ia : Sp ;=9ˏG:C-C:G f

ZmY*ʫ>÷ȿŪˏ
Ȝ2Ț

'

>

@



;/Ȝ 3 Ț&, Oz *ĺ%õ=ʎ5?>Ěė'%REi*# %+6ɧò)<DNA z~] Oz *ĺ%õ=ʎ7Ěė,REi&,) ıʳ*¥Ü+ȊȖȱƭ 20, 21);/Ȝ 4 Ț+ɥƫȱƭ <KF exo-

Pol γPol κPol IV , Oz *ĺ%REi'Eei+ƕBPol η *#%,RE

XfX9õ=ʎ6Pol ζ : Pol ι *ɉ"%,REiEei

i'Eei*ßXfXb|+É%Bõ=ʎ7'ƛ<
DNA z

;/

~]*

')"%

>#5=?<+%,Oz *ĺ%REiõ=ʎ5?Ś+©ʨóŦE

ei:XfXb|õ=ʎ5?ęù*ǂ2%ãǪǼ*ɕA?>ęù+6
G:C-C:G fZmYƤŮ*őʂ?>'ȺɌǼ*|Z{cbĚėĺ&>t
ːq

ːt

|YĚėĺ (Pyr:Pyr) , Pur:Pyr'A?%Ěėĺ (Pur:Pur)

;/q

|Y

;/ Pyr:Pur |Z{cbĚėĺ;=9 DNAƽ6:ĭİ&=DNA ɜɛBżDNA Bɜɛ*

<?>

DNA z

~],+;)ƽC>ÕĨäÞĪX|X*;"%tG{

Ƨț*|Z{cbĚėĺ (G:G 5, C:CC:A Ěėĺ) Bü7 DNA ,ƽ7'
%+ƽ6Ŏù

, G:G > C:C > C:A &>'?5&*ȍ?%> 86)5¥Ü Beard <,tG{Ƨț Pur:Pyr 5, Pyr:Pur Ěėĺ+ƕ Pur:Pur
78

;/ Pyr:Pyr Ěėĺ;=9Ƽȋ)Ěė+õ=ʎ6ãǪˏ'BĘþ%> 87)

+8DNA z~]+©ʨóŦ+ãǪ,tG{Ƨț+Ěėĺ+ĭİŪ*²

ĩ%

>

>'

?5&+ȊȖȱƭ 20, 21) *ßȜ 4 Ț+ɥƫȱƭ<REV1 BʰÉ%+ DNA~]Ëʑ% Oz *ĺ%REiBõ=ʎ7'ƛ<

z

')"%

=

Oz ' G  A : CT ;=9ĭİ)ĚėĺBŕų>'ȍĄ?5ʤĨçĪɨȠ*;"% Oz:G Ěėĺ Oz:A Ěėĺ;=9ĭİ)ĚėĺBŕų>'ǖ?%>'<9 20)Oz *ĺ%REiõ=ʎ5?Ś+©ʨóŦ,Eeiõ=ʎ5?ęù;=9ãǪǼ*ɕA?>'ǖ?>&ƨȚ&,ıʳ+
DNA ©ʨóŦ*

> Oz:GĚėĺƣ9ĭİ&>(

Ț&,tG{©ʨóŦBǰ;/ Oz:AOz:COz:T Ěėĺ+ĭİŪBɫ³Oz:G
Bƛ<*>'Bɬ6+ȀǼ+8*ƨ%tG{Ƨț+©ʨóŦWGf* Oz:G 5,

Oz:AOz:COz:T ĚėĺB9#tG{+©ʨãǪ+ʙ
5&+ Oz *ĺ>Ěė+õ=ʎ6ɥƫ*

>ȱƭ'+ɻŪBɫ³>8*Oz*ĺ>Ěė+õ=ʎ6n`ǵ)> 3 #+ DNA z
õ=ʎ7 Pol β~] (REi+6B;/REi'EeiBõ=ʎ7 KF exo-REi'EeiX

fXBõ=ʎ7 Pol η) Bǰƨʧ+ĭİŪ

Bǂʊ5?

<*ǤĞŪ*;= Tm ºBǖİ>'&DNA;/ƨʧ*ü5?>Ěėĺ+ĭİŪBǖİ

79

Ȝ 2 ȡ ıˋƕǈ2-1. L

VjQLbgDNA z~]*;>tG{©ʨóŦ*ǰ 30-mer + DNA dtf(5'-CTCATCAACATCTTXAATTCACAATCAATA-3'X = Oz)
 9-mer + DNA L;/ǤĞŪıˋ*ǰ

V{ (5'-TGCTXGCGT-3'X = Oz) ,¥Ü*ʐ2<?; * 20)ɵɛtG{©ʨóŦ+Uf*ǰ
(5'-CTCATCAACATCTTGAATTCACAATCAATA-3')

 DNA d  t   f;/ 5'Ƨț+ Alexa680 vçtG{ (5'-TATTGATTGTGAATTN-3'N = C 5, GAT) ,ƘƨmGLWp
Z;=ɿÈǤĞŪıˋ*ǰ
5, T
Bǰ 9-mer DNA L

V{ (5'-TGCTNGCGT-3'N = C;/ 5'-ACGCNAGCA-3'N = C 5, GAT) ,xZx|aGfǈ%ùų

2-2. DNA z~]

KF exo-, Fermentas (Waltham, MA)<hPol β , CHIMERx (Milwaukee, WI)

<ɿÈ


hPol η , POLH *;"%Ug?%=+ÕĨʤ, 78 kDa &>N Ƨț*

6×His `Rɔù hPol η BUg>ġɄɐǻǫǰvQ`pET15b/hPol η BƵȢ (Fig.40)pET15b/hPol η Bȯ6ʎC ġɄɐ Rosetta2 (DE3) pLysS * 1 mM IPTG Bß%ǻǫɰĽǻǫɰĽġɄɐ, 37 ºC & 3 ƝʪĖˉŚʚţÕʷ*;=ċ

ô-80 ºC *Òȱµĩ¥ʮ+Ɗ®,Ȝ 4 Ț+ hPol δ 'úƶ*ɕhPol η µĩǰmcrD (20 mM sodium phosphate (pH 7.4), 0.1 M NaCl, 1 mM EDTA, 10% glycerol,
10 mM 2-ME, 0.5 mM PMSF) *ȷƇĿʤ#ÕǊ%-80 ºC *µĩ

80

Fig.40 Expression and purification of hPol η.

2-3. CBB ƮɎ®ɛ hPol η +Uphf*ǘɥmcrD (62.5 mM Tris-HCl (pH 6.8), 10%

glycerol, 2% SDS, 0.05 mg/ml bromophenol blue) Bß5 Õʪ 95 ºC &Óǭ+Ś,Ȝ 4 Ț'úƶ+ƕǈ&ɕ"

2-4. HIZ`scdFRǈ
HIZ`scdFRǈ,Ȝ 4 Ț'úƶ+ƕǈ&ɕ"1 ƺŷ­'%
Anti-His-tag (MBL) B2 ƺŷ­'% Peroxidase-conjugated AffiniPure Goat Anti-Mouse
IgG (H+L) (Jackson ImmunoResearch) B¯ǰ81

2-5. tG{©ʨóŦ
KF exo-;/ hPol βhPol η + DNA z~]óŦɥƫ*#%ƺ*ȍ
KF exo-+óŦǐ+ȯų, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 100 µg/ml BSAˑhPol
β +óŦǐ+ȯų, 50 mM Tris-HCl (pH 8.8), 10 mM MgCl2, 1 mM DTT, 400 µg/ml BSAˑhPol η +óŦǐ+ȯų, 50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 5 mM DTT, 100 µg/mL
BSA &>KF exo-;/ hPol βhPol η +óŦǐ, 100 fmol + DNA dtf;/ 50 fmol + 5'ƧțB Alexa680 &ƷɹçtG{100 µM dNTPs (dCTPdGTP
dATP;/ dTTP) BüC&

>?? DNA z~]+ǜŎ,??+ď+ɳƛƓ*ɪʋ5tG{©ʨóŦ*ǰ DNA dtf't

G{+ȯ6ùA, Fig.41 *ȍ¥Bü7óŦǐ,ÉʤB 5 µl 'KF exo;/ hPol βhPol η +óŦǐB 37 ºC & 30 ÕʪóŦ5 µl + stop mcrDBß%óŦB¼ƻ%8 M ŀȫBü7 16%z

EQ

mcrDBǰ

! 2.5 µl B 30 W & 60 ÕʪʹǃǋäBɕ%??+óŦŚ+Wt+E|gT' 1×TBE

ÕʷʹǃǋäŚÕʷT, Odyssey® Infrared Imaging System(LI-COR Biosciences) B¯ǰ%ɥƫ
5©ʨãǪ,Ő (14) +;*Éʨ5&©ʨmg+œŎ (Bandfull) Bt

G{Bü7mgÉ­+œŎ (Bandtotal) &Ý"ǂǪ'%İȸȠÔ

Fig.41 Experimental design of DNA polymerase assay for primer extension. The 16-mer primer
is labeled with Alexa 680 indicated by ‘*’.
82

Extension!efficiency!(%) =Band!"##
×100
Band!"!#$(14)

2-6. G 5, Oz + 5'½ʴƄĚė*ĺ>Ěėõ=ʎ6ɥƫ
G 5, Oz *ʴƄ>Ěė*ĺ>Ěė+õ=ʎ6*ʫ>ĚėʜŹŪ+Õƫ*ǰóŦǐ+ȯų,tG{' dNTP ¥ğ,tG{©ʨóŦ'ú&>20
nM DNA dtf G +ęù, 10 nM tG{5'-TATTGATTGTGAATTAC-3'
Oz +ęù, 10 nM tG{5'-TATTGATTGTGAATTAG-3'Bǰɥƫ*ǰĚė+õ=ʎ6

 DNA dtf'tG{+ȯ6ùA, Fig.42 *ȍ5

?+óŦǐ*, 100 µM + dNTP ë­ (dCTP 5, dGTPdATPdTTP) Bǰ

Fig.42 Experimental design of DNA polymerase assay for incorporation opposite 5'-neighboringbases of G or Oz. The 16-mer primer is labeled with Alexa 680 indicated by ‘*’.

83

2-7. ǤĞŪıˋ
Tm ºBǇİ>8* 9-mer + DNA (5'-TGCTXGCGT-3'X = C 5, TOz) ' 9-mer+Ȃɚʧ (5'-ACGCNAGCA-3'N = G 5, ACT) Bǰ%ǤĞŪıˋBɕ"

50 mM sodium phosphate (pH 7.0)1 M NaCl *9-mer + DNA ' 9-mer +Ȃɚʧ+ǂ, 1:1 &ƨʧ+ȮǜŎ 4 µM ')>;

V{,60 ºC & 5 min ßǤŚĲǕ5&??+Wt*ü5?>ȂɚǼ)Lś*Ðî>Ei*Wt (100 µl) BɵƑ

R*;"%ƨʧBŕųÉ%+Wt,ßǤ*;>ɒǻBʬ8*90 µl +X

ULG (Life Technologies) &ɞ"%

Ultrospec 3100 pro (GE Healthcare) Bǰ %Ei
Z& 20 ºC

RŚ+WtB 1 ºC/min +w< 80 ºC 5&ßǤ260 nm &ýÆŎBi`

Tm º,ɔɥƢȵ+ƺŠÕ+ƣġº<Ǉİ

84

R??+Ȝ 3 ȡ ȱƭ'ȺĹ
3-1. hPol β +tG{©ʨóŦOz *ĺ%REi+6Bõ=ʎ7 hPol β +ƈ¿Bʄ
+ɥƫ*,30 Ěė%©ʨ>ãǪBɵ2<)> G 5, Oz BĚėü7 DNA dtf;/

G 5, Oz +Ȃɚʧ½*>tG{+ 3'ƧțĚė??ǵ)> 16 Ěė)> 4 ȓˈ+tG{Bǰ<

G Bü7 DNA dtf+ęùhPol β ,tG{+ 3'ƧțĚė C +tG{<+ƣ9ãǪɍ©ʨ+¢+tG{Ƨț*|Z{cbĚėĺB?

?ŕų>¢+ 3 #+tG{+©ʨãǪ,¬5, 710 Bǂʊ)#5=f^Q&tG{Ƨț*©ʨ%" (Fig.43A + 1 ' 4cQĚėĺƣ9ĭİ&>'Bȋɯ

> G:T |Z{cb,¢+ 2 #+|Z{cb;=,ãǪɍ (Fig.43A + 10 ' 4 5, 7 Bǂʊ;/ Fig.43C)

?<, Pur:Pyr Ěėĺ Pur:Pur Ěėĺ;=9ĭİ&>ʘð+Ęþ 87) 'Ɋ%ƕOz Bü7 DNA dtf&,Oz +Ȃɚʧ½ G ')>tG{<ƣ9ãǪɍ©ʨ%= (Fig.43B + 4 ' 1 5, 710 Bǂʊ)¢+ęù+Ŀ)'9 8 ¹ãǪǼ*Éʨ5&©ʨ%tG{Bǰ

?<+ȱƭ, Oz *ĺ%REi+6Bõ=ʎ7'

 (Fig.43D)

ʘð+ıˋȱƭ 20) 'Ɋ?ƎhPol β Oz *ĺ>REi+õ=ʎ6, Oz:G Ěėĺ+ĭİŪ*ʂČ%%>'ƅǖ

<*Oz:G<Éʨ5&+©ʨ, G:G : G:A |Z{cb<+©ʨ;=9ãǪɍ" (Fig.43B + 4 ' 43A + 4 5, 7 Bǂʊ)?<,hPol β +;)ŧıŎ+ˏ

DNA z~]*;> DNA ɜɛʘȒ*

Pur:Pur |Z{cbĚėĺ;=9ĭİ&>'Bȍ

85%Oz:G Ěėĺ

Fig.43 Extension from primer ends by hPol β. Each primer contained a different nucleotide atthe 3' end (indicated by N, where N was C, G, A, or T in lanes 1-3, 4-6, 7-9, and 8-12,
respectively) opposite G (A) or Oz (B). The amount of hPol β was 25 mU in lanes 1, 4, 7, and 10
or 2.5 mU in lanes 2, 5, 8 and 11. The extension efficiency beyond G:C, G:G, G:A or G:T (C),and Oz:C, Oz:G, Oz:A or Oz:T (D). Reproduced from Suzuki M. et al., J. Nucleic Acid, 2014,
178350, (2014) with permission from the Hindawi Publishing Corporation.
86

3-2. KF exo-+tG{©ʨóŦ
ƺ*KF exo-+ǍŪ*#%Ƴɩ (Fig.44)KF exo-, hPol β 'úƶ*tG{Ƨț G:C Ěėĺ')>tG{+©ʨãǪ¢+Ěėĺ+ęù*ǂ2%ɍ
 (Fig.44A + 1 ' 4 5, 710 Bǂʊ)?*ß
ʨãǪɍ

"

%tG{+©

" 3'Ƨț*«ȷ>Ěėĺ+˃«, hPol β 'úƶ* G:C > G:T > G:A >G:G &" (Fig.44A, C)
KF exo-, Oz *ĺ%REi'Eei+ƕBõ=ʎ7 20)tG{+©ʨãǪ&,ˌ2'*3'Ƨț Oz:G Ěėĺ')>tG{" (Fig.44B + 4)<*Oz:G Ěėĺɍ

Oz:T Ěėĺ

<+©ʨ;=9 2 ¹¥ˏ<+©ʨãǪ, Oz:C : Oz:A" (Fig.44B + 4 ' 1 5, 7

10 Bǂʊ ;/ Fig.44D)?,tG{©ʨóŦ*
Ěėĺ;=9ĭİ&>'Bȍ%<+©ʨãǪƣ9%Oz:G Ěėĺ Oz:A

=+Ěėĺ+ĭİŪ+ʙ

Ƞ*;> Oz:G Ěėĺ Oz:A Ěėĺ;=9ĭİ&>'87,ʤĨçĪɨ¥Ü+ǖ 20) 'Ɋ%Fig.44 Extension from primer ends by KF exo-. Each primer contained a different nucleotide atthe 3' end (indicated by N, where N was C, G, A, or T in lanes 1-3, 4-6, 7-9, and 8-12,
respectively) opposite G (A) or Oz (B). The amount of KF exo- was 250 mU in lanes 1, 4, 7, and10 or 25 mU in lanes 2, 5, 8, and 11. The extension efficiency beyond G:C, G:G, G:A or G:T (C),and Oz:C, Oz:G, Oz:A or Oz:T (D). Reproduced from Suzuki M. et al., J. Nucleic Acid, 2014,
178350, (2014) with permission from the Hindawi Publishing Corporation.
88

3-3. hPol η +tG{©ʨóŦ
hPol β : KF exo-'úƶ*hPol η *;> G 5, Oz +Ś+tG{©ʨãǪBɥƫhPol η *#äŚ+ CBB ƮɎ

%,UphfB®ɛz

EQ

E|gTʹǃǋ;/HIZ`scdFRǈ*%ȋɯ (Fig.45)hPol η 9

hPol βKF exo-'úƶ*tG{+ 3'Ƨț G:C Ěėĺ+ęùƣ9ãǪɍ©ʨ (Fig.46A + 1 ' 4 5, 710 Bǂʊ)'@hPol β : KF

%exo-',ʙ"%tG{Ƨț*|Z{cbĚėĺ (G:G 5, G:AG:T) Bü7ęù&+©ʨãǪ, 50%¥&" (Fig.46A + 4

Fig.46C)+ȱƭ,hPol η Ʀƈ¿+ DNA ɜɛ*
88-90)'Ɋ%;/ 710 ;/%ɲ=!&>'Ęþ

hPol η , Oz *ĺ%REi'EeiXfXBúȒŎõ=ʎ7'Ɨ*ƛ<*)"%= 20)<* hPol η ,ɲ=!) DNA ùųBɕ

&>'Ȉ<?%

>*9

DNA z

~]

A< 88-90)Oz:G ĚėĺB 3'Ƨț*ü7tG{, Oz:C : Oz:AOz:T Bü7tG{+©ʨ;=9ȧ 2 ¹ãǪǼ&" (Fig.46B+ 4 ' 1 5, 710 Bǂʊ;/ Fig.46D)+ȱƭ,Oz:G Ěėĺ¢+Ěėĺ;=9ĭİ&>'Bȍ%=hPol β : KF exo-+ȱƭ'Ɋ%

Fig.45 Expression and purification of recombinant hPol η. (A) hPol η was electrophoresed andstained with CBB. (B) hPol η was identified by immunoblotting with anti-His antibody.
89

Fig.46 Extension from primer ends by hPol η. Each primer contained a different nucleotide atthe 3' end (indicated by N, where N was C, G, A, or T in lanes 1-3, 4-6, 7-9, and 8-12,
respectively) opposite G (A) or Oz (B). The amount of hPol η was 11.5 ng in lanes 1, 4, 7, and10 or 1.15 ng in lanes 2, 5, 8, and 11. The extension efficiency beyond G:C, G:G, G:A (C) or
G:T, and Oz:C, Oz:G, Oz:A or Oz:T (D). Reproduced from Suzuki M. et al., J. Nucleic Acid,
2014, 178350, (2014) with permission from the Hindawi Publishing Corporation.
90

3-4. Oz Bü7 DNA ƨʧ+ǤĭİŪ
<*Oz:G 5, Oz:AOz:COz:T ĚėĺBü7 DNA ƨʧ+ Tm ºBǤĞŪı
ˋ*;=ɥƫ

DNA z~]*;>tG{©ʨóŦ*ǰ

VjQLbg+ʤ,ǤĞŪıˋBɕ
bg+ùų&,L;/ 3-23-3 **,èÕ&"Oz Bü7L

VjQLbg+ʨȉ

L

VjQL

ȒôʤĜß>+83-1

>tG{©ʨóŦ',ǵ)"ȉʠÖBǤĞŪıˋ*ǰ>'*DNA ƨʧ+ Tm º+ǖİȱƭ<C:G ĚėĺBü7ęù, 55.1 ºC &= (Table 10)(Fig.47A ' 47B)A:T Ěėĺ&, 48.9 ºC &" (Table 10) (Fig.47C ' 47D)?,
C:G Ěėĺ A:T Ěėĺ;=9ǤÞĪǼ*ĭİ&>''Bȍ%

Ɍ*??+ĚėĺBŕų>Ǆȫȱù+Ɛ+ŉ*ʂČ>'=?,

èÕ*ȋș

?ı&>
Oz:G ĚėĺBü7 DNA ƨʧ+ Tm º, 45.7 ºC &" (Table 10) (Fig.47E ' 47F)
ƕOz:A 5, Oz:COz:T ĚėĺBü7 DNA ƨʧ+ Tm º,ƛ<*ƳÔʯǳ¥ (Tm º < 40.0 ºC) &"8 (Table 10) (Fig.47G-L)Tm ºBǇİ&)"#5=Oz:G Ěėĺ,¢+ Oz:A : Oz:COz:T Ěėĺ;=9ƤŮ*ǤÞĪǼ*ĭİ&"

Table 10 The Tm values of C:G, A:T, Oz:G, Oz:A, Oz:C and Oz:T.
base pair

Tm value (ºC)

C:G

55.1

A:T

48.9

Oz:G

45.7

Oz:A

< 40.0Oz:C< 40.0Oz:T< 40.091

Fig.47 Cont.

92

Fig.47 Cont.

Fig.47 Melting curves of (A) C:G, (C) T:A, (E) Oz:G, (G) Oz:A, (I) Oz:C and (K) Oz:T 9-mer
DNA duplexes at 4 µM duplex concentration. The first derivative of the melting curve of (B)C:G, (D) T:A, (F) Oz:G, (H) Oz:A, (J) Oz:C and (L) Oz:T. Y-axis is the first derivative ofabsorbance at 260 nm with respect to temperature. Reproduced from Suzuki M. et al., J. Nucleic
Acid, 2014, 178350, (2014) with permission from the Hindawi Publishing Corporation.
93

3-5. G 5, Oz + 5'½ʴƄĚė*ĺ>Ěėõ=ʎ6ɥƫ<hPol β : KF exo-hPol η ,tG{+Ƨț Oz:AtG{©ʨóŦ+ɥƫ: Oz:COz:T Ěėĺ;=9 Oz:G Ěėĺ+ƕãǪɍ©ʨ:ƈ¿=ʄ

DNA ùų*%Ƽ'Bƛ< *ĚėƂÈ?%>'ʣ

ɝ&>&hPol β : KF exo-hPol η  Oz:G ĚėĺŕųŚ+ DNA ɜɛʘȒ*%ĚėBƼȋ*õ=ʎ7(

Bƛ<*>8*G : Oz + 5'½ʴƄĚė*ĺ>Ěė+õ=ʎ6ɥƫBɕ"DNA dtf'tG{*#tG{©ʨóŦ*%ƣ9ãǪɍ©ʨtG{+Ƨț G:C 5,Oz:G Ěėĺ')>ȯ6ùABǰ

G Bü7 DNA dtf+ęùhPol βĺ%dATP Bõ=ʎC%,;/ KF exo-, G *ʴƄ> 3'-TT-5'*(Fig.48A ' 48B + 4)G *ʴƄ> T *ĺ

> dGTP +ɲõ=ʎ69Ŀ6<? (Fig.48A ' 48B + 3)ƕhPol η ,hPol β : KF exo-',ǵ)=3'-TTCT-5'*ĺ% dATP õ=ʎ5?%
G *ʴƄ> T *ĺ% dGTP ' dTTP 9õ=ʎ5?%?, hPol η +ǦŢ&>ɲ=!)ɜɛBɕ<?>;/ KF exo-, Oz *ʴƄ> (Fig.48A ' 48B + 9)G Bü7 DNA dtf+ęù*ǂ2%+ãǪ,¬tf+ęù;=9ãǪ¬ (Fig.48C + 3-5)

ǍŪ 88-90) *;"%ǯ'Ⱥ

 Oz Bü7 DNA dtf+ęù&9hPol β3'-TT-5 *ĺ%dATP Bõ=ʎC'*ß"hPol η *#"õ=ʎC%9 G Bü7 DNA dĚė+ȓˈ, G Bü7 DNA dtf+ęù'úƶ* 3'-TTCT-5'*ĺ% dATP Oz *ʴƄ> T *ĺ% dGTP
' dTTP õ=ʎ5?%

 (Fig.48C + 8-10)?*ßhPol β;/ KF exo-',ǵ)"% hPol η ,Oz *ʴƄ> T *ĺ> dCTP +õ=ʎ69ɟ<? (Fig.48C+ 7)¥<hPol β,Ƽ Ěėõ=ʎ5?%+ęù+6;/ KF exo-, Oz B=ʄŚ+©ʨóŦ*%?*ĺ%hPol η , DNA dtf Oz&)G +ęù&9ɲ=!)ɜɛɕA?%94



Fig.48 Nucleotide incorporation opposite the bases adjacent to G or Oz by hPol β (A), KF exo(B), or hPol η (C). Left panels of (A), (B), and (C) show the control, which was extension ofprimers containing C opposite an undamaged G in the template. Right panels of (A), (B), and (C)show the extension of primers containing G opposite Oz in the template. The amount of hPol β
or hPol η was 25 mU or 11.5 ng; the amount of KF exo- was 25 mU for the left panel, or 250
mU for the right panel. Reproduced from Suzuki M. et al., J. Nucleic Acid, 2014, 178350,(2014) with permission from the Hindawi Publishing Corporation.

95

Ȝ 4 ȡ ȱɶ¥Ü+ȊȖȱƭ 20, 21);/Ȝ 4 Ț+ɥƫȱƭ* Oz *ĺ%õ=ʎ7Ěė, DNA z<DNA z~] dGTP ¥ğ~]'*ǵ)"%>'ƛ<')"5Ȝ 2 Ț*ȍ; *ʤĨçĪɨȠ*;"%Oz ,REi'ĭİ)Ě
ėĺBŕų>'ǖ?ƨȚ&,Pol βz~]óŦɥƫėĺʪ+ĭİŪ+ʙPol β<DNA ©ʨóŦ*Bƛ<;/ KF exo-Pol η Bǰ

>Oz:G ' Oz:AOz:COz:T Ě*>'Bɬ6

;/ KF exo-Pol η *Ëʑ%Oz:G ĚėĺBʄOz:C : Oz:AOz:T +ęù;=9ˏ%©ʨ>ãǪ+ƕ<*Oz:G 5, Oz:C'Bɟ

Oz:AOz:T ĚėĺB 1 #ü7ƨʧ+ Tm º<ǤÞĪǼ)ĭİŪ*Ěėĺ, Oz:C : Oz:AOz:T Ěėĺ;=9ƤŮ*ˏ 'ƛ<¥;=DNA z DNA~]óŦ

')";/ǤĞŪ*ʫ>ıˋ*)Ěėĺ&>'Bȍ?<+ȱƭ,¥Ü+ʤĨçĪɨȠǖ 20) 'Ɋ%ėĺ Oz:A Ěėĺ;=9ĭİ&>'%9 Oz:G%Oz:G ĭİ<ŝ Oz:G Ě#5=ƨȊȖȱƭ,G:C-C:G fZmY*ʫ>REiʢçƈ¿'%+ Oz +ʣɝŪ
B;=œç>9+&> ŚOz:G Ěėĺ+ƵʓɥƫɕA?>'&;=ɮȭ* Oz:G Ěėĺ+ĭİŪƛ< *)>

@

96



Ȝ6Ț
Oz ʔȳʠÖ+ DNA z ~]óŦ*ĺ>Ŗʿ(ƨȚ+ÍĶ*ʫ%, J. Biochem., 159, 323-329 (2016) *ǻɘ6)

Ȝ 1 ȡ Ƚƞ
ÜȚ5&,1 #+ Oz *ȅȀĚė+õ=ʎ6:=ʄ

DNA ùų*#%ɥƫ

%REi,ʔȳʠÖ'%ĩđ>ęùREië­&ĩđ>ęù;=9ʢçʹ«¬ʹĨʢç*;"%ʢç?:)> 91, 92)5REiʔȳʠÖ+REi,ʹĨʢç<')"%&)ʣ˂ʢȫ*;"%9ʢç?>'ƛ> 93)#5=ˏʢçƩ¦*%ʢç?ȳęùREi

<REiʢçƈ¿+ʔȳʠÖǯų>+REi+ʔȳʠÖ, K-rasʔȳʠÖʝ¨Ĩ:d~EʠÖ'"ʣɝ)Tl}˄Ĕ*9ĩđ%=DNA ɜɛʘȒ*

>REiʢçƈ¿+ʔȳʠÖ+ŖʿBɥƫ>'Ťɝ'?%
DNA ɜɛʘȒ*

> 8-oxoG +ʔȳʠÖ+ŖʿBɥƫĘþ=8-oxoG ʔȳʠÖ, Pol α : Pol β + DNA ùųBʭĴ>'ƛ<ȳʠÖ (5'-IzIz-3') ǯų>'Ɨ*ƛ<?% Oz Bǯų>'')"%< 11)+ Iz ʔȳʠÖ*ǯų>',Ɉƛ&>5Iz ,REi
9ǯų>'9Ⱥű>'Oz ë­'Ⱥ> 94, 95);/ʘð+Ęþ

"ˏŧıŎB9" DNA z

Bùų&>'ƛ<;> 13)Iz ,ś*ßǄÕɥ< Oz ʔȳʠÖ (5'-OzOz-3') ĶƜ&) 8-oxoG +ʢç*;"%&) Oz ʔȳʠÖ+ǯǥĪǼ)Ŗʿ,ǟɠ

<?>

Ȝ4Ț

ʭĴ)

')"%

;/ƨʧ DNA +REiʔȳʠÖ (5'-GG-3') Bʢçʳ*Iz ʔ

ƨʧ

&)

>ıʳ*

<DNA ɜɛ+ţǼŗÝBż')"%Pol α : Pol δPol ε '~]&9 Oz  1 #+ęù,=ʄ
>

% DNA

)<ë­+ęù*, DNA ùųB) Oz &9ʔȳʠÖ'%ǯųęù,Oz  1 #+ęù'ǂ2>' DNA ùųBʭĴ:)>;ƨȚ&, KF exo-)ʣȣ)ƈ¿')>'ǖ?>&;/ calf thymus Pol (ctPol) αhPol βyPol ζhPol ηhPol ιhPol κ
97yREV1  Oz ʔȳʠÖB=ʄ
?<+ DNA z% DNA Bùų&>(Bɥƫ5~] Oz ʔȳʠÖ*ĺ%ĚėBõ=ʎCȓˈBɥƫ?5&+ Oz *ʫ>Ěė+õ=ʎ6ɥƫùųóŦɥƫ

<DNA zʿ)'Ɨ*ƛ<]Bǰø DNA z~]ofǱƪ ʡǁǱƪ')"%ęù,+Ěė+;/ƈ¿=ʄ(' "ȓŉŖ8ƨȚ&,ɪ*ȍ DNA z~]+Ƹȿ*# %, Table 11 *ȍ

Table 11 The function of DNA polymerases used in this chapter 42, 43).
DNA polymerase
Pol αReplicative DNA polymerases

Pol β

DNA polymerases

KF exo-involved in DNA repair

Pol ζ
Pol η
Pol ι

DNA polymerases
involved in translesion synthesis

Pol κ
REV1

98

DNA

~

Ȝ 2 ȡ ıˋƕǈ2-1. L

VjQLbg

DNA z~]óŦ*ǰ Oz ʔȳʠÖBü7 30-mer +L

VjQLbg(5'-CTCATCAACATCTXXAATTCACAATCAATA-3'XX = OzOz) , 2 #+ǀʲ*Õ%ɵɛ (Fig.49)5ʘð+Ɠǩ 13) BñȺ*REiʔȳʠÖBü7 6-mer +L
VjQLbg (5'-CTGGAA-3')<

ʢç*;= Iz ʔȳʠÖBü7 6-mer +L

yrpĩđ&+ UV (365 nm) ǢĻ+Æ
VjQLbg (5'-CTIzIzAA-3') Bɵɛ

+Ś65 ºC & 75 ÕʪßǤ>'& Oz ʔȳʠÖBü7 6-mer +L

VjQLbg (5'-CTOzOzAA-3') 1'ßǄÕɥ 11)ƺ*Oz ʔȳʠÖBü7 6-mer +L;/ 13-mer L

QLbg

VjQLbg (5'-TTCACAATCAATA-3') B T4 z

jQLbgOh] (New England Biolabs, Ipswich, MA) Bǰ%Oz ʔȳʠÖBü7 6-mer +L% 5'ƧțBʢç

VjQLbg+ 5'½* 11-mer L

jQLbg (5'-CTCATCAACAT-3') B3'½*
gB T4 DNA

Vjʢç 13-mer LN] (Takara Bio, Otsu, Japan) BǰV

VjQLb%GTX+ʳ

d  t   f '  % 30-mer DNA-RNA O ~  L

V j Q  L b g(5'-TATTGATTgTGAATTGCAGATgTTGATGAG-3', “g” , 2'-eLOXRElX&,)y­+RElX) BǰLbg, HPLC BǰƣȮǼ*Oz ʔȳʠÖBü7 30-mer L

VjQ%ëʷtime-of-flight mass spectrometer (Bruker Daltonics K.K.,

Yokohama, Japan)*;=ÕĨʤ< Oz ʔȳʠÖBü7 30-mer L

VjQLbg&>'Bȋɯ
DNA z~]óŦ+Uf'%ǰ

REiʔȳʠÖ5, CPD B

ü7 30-mer +L

VjQLbg (XX = GG 5, CPD)Ʒɹç?L

VjQLbg(5'-*TATTGATTGTGAATT-3') ,ƘƨmGLWpZ;=ɿÈ99;/ 5'ƧțB Alexa680 &Fig.49 Outline of the preparation of 30-mer DNA containing the OzOz lesion. XX represents
OzOz. “g” represents guanosine, not deoxyguanosine. Reproduced from Suzuki M. et al., J.
Biochem., 159, 323-329 (2016) with permission from the Oxford University Press.

2-2. DNA z~]

KF exo-, Fermentas
yREV1 , Enzymax<ctPol α

yPol ζ

<yPol ζ;/<ɿÈ5hPol η ,Ȝ 5 Ț'hPol ι

2-3. DNA z;/ hPol β , CHIMERx;/ hPol κ ,Ȝ 4 Ț'úƶ+ƕǈ&®ɛ~]óŦ

;/ hPol ιhPol κyREV1 + DNA z;/ hPol βhPol η + DNA z
ctPol α + DNA z~]óŦ,Ȝ 4 Ț'KF exo-

~]óŦ,Ȝ 5 Ț'úƶ+ƕǈ&ɥƫ5~]óŦɥƫ*#100%ƺ*ȍctPol α +óŦǐ+ȯų, 40 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 10 mM NaCl, 45 mM KCl, 10 mM DTT, 250 µg/ml
BSA, 5% glycerol &>ctPol α +óŦǐ, 100 fmol + DNA dtf+ 5'ƧțB Alexa680 &ƷɹçtG{BüC&
DNA z
óŦ

;/ 50 fmol>??+ dNTP;/~]+ǜŎ,??+ď+ɳƛƓ*ɪʋ5tG{©ʨ;/Ěė+õ=ʎ6ɥƫ*ǰ

 DNA dtf'tG{;/ dNTP+ȯ6ùA, Fig.50 *ȍ¥Bü7óŦǐ,ÉʤB 5 µl 'ctPol α +óŦǐB 37 ºC & 30 ÕʪóŦ5 µl + stop mcrDBß
8 M ŀȫBü7 16%z
óŦŚ+Wt+EQ%óŦB¼ƻ%

E|gT' 1×TBE mcrDBǰ! 2.5 µl B 30 W & 90 ÕʪʹǃǋäBɕ%??+

ÕʷʹǃǋäŚÕʷT, Odyssey® Infrared Imaging System (LI-COR Biosciences) B¯ǰ%ɥƫ

Fig.50 Experimental design of DNA polymerase assay. The 15-mer primer is labelled with
Alexa 680 indicated by ‘*’.

101

Ȝ 3 ȡ ȱƭ'ȺĹ
3-1. Oz ʔȳʠÖBü7 30-mer + DNA dtf+ɵɛ
5ʘð+Ɠǩ 13) BñȺ*REiʔȳʠÖBü7 6-mer +L

VjQLbg< Iz ʔȳʠÖBü7 6-mer +L

VjQLbgBɵɛŚ (Fig.51A)ǤÕɥ*;= Oz ʔȳʠÖBü7 6-mer +L

VjQLbgBŝ (Fig.51B)Oz ʔȳʠÖBü

7 6-mer +L+L

VjQLbg+ôǪ, 1.3%&"+ŚOz ʔȳʠÖBü7 6-mer

VjQLbg+ 5'½* 11-mer3'½* 13-mer +L

X>'&Oz ʔȳʠÖBü7 30-mer +L
DNA z~]óŦɥƫ*ǰ

6-mer +L

VjQLbgBGT

VjQLbgB®ɛ (Fig.48C)

5Ôǻǥɾ&>REiʔȳʠÖBü7

VjQLbg <6 Oz ʔȳʠÖBü7 30-mer +L

VjQLbgôǪ,0.12%&"
Oz ʔȳʠÖBü7 6-mer +L

VjQLbg+ 5'½* 11-mer3'½* 13-mer +L

VjQLbgBʔȱ*,30-mer + DNA-RNA O~LŔ×30-mer + DNAf'%ǰ
f'%ǰ

<)>L

VjQLbgB DNA dtʔȱŚ+ Oz ʔȳʠÖBü7 30-mer +L+ DNA dtfB HPLC &,Õʷ&)
jQLbg+ʟ* RNA Bü7O~L

VjQLbg'"&dtf+L
VjQLbgBǰ

@HPLC *% Oz ʔȳʠÖBü7 30-mer +Lƛ<VjQLbgBdt%ÕʷBɬ6'VjQLbg'Õʷ&>'*)" (Fig. 51C)+8ƨȊȖ&, 30-mer + DNA-RNA O~L

QLbgB DNA ʔȱƝ+dtf'%ǰ

102

V



Vj

Fig.51 HPLC analysis for preparation of 30-mer DNA template containing the OzOz lesion.
HPLC isolated (A) 6-mer-IzIz, (B) 6-mer-OzOz, or (C) 30-mer-OzOz. Sample analyzed by
HPLC with a CHEMCOBOND 5-ODS-H columun (Chemcoplus, Osaka, Japan, 5 mm, 150 ×
4.6 mm, elution with a solvent mixture of 50 mM TEAA (pH 7), (A, B) 6.2-7.2 % during 0-30
min, (C) 10-10.5% during 0-30 min, CH3CN at a flow rate of 1.0 ml min-1) and monitored at 260
nm absorbance.

1033-2. KF exoKF exo-;/ ctPol αhPol β + Oz ʔȳʠÖ*ĺ> DNA z;/ ctPol αhPol β  Oz ʔȳʠÖB=ʄɥƫUf'%ǰ
ęù,KF exo-

~]óŦɥƫ% DNA Bùų&>REiʔȳʠÖBü7 DNA dtf+;/ ctPol αhPol β ,ãǪɍÉʨ5&©ʨ% (Fig.52A-C + 1-3)ƕOz ʔȳʠÖBü7 DNA dtf+ęù,KF exo-+Ś+©ʨóŦ,¼ƻ%
ƺ*KF exo-;/ ctPol%3'½+ Oz *ĺ> 1 Ěė+õ=ʎ6+6ƳÔ?%αhPol β É%*(= (Fig.52A-C + 5)

;/ ctPol αhPol β  3'½+ Oz *ĺ%õ=ʎC

1 ĚėBɥƫ+ȱƭKF exo-, 3'½+ Oz *ĺ% dATP BÂ«*õ=ʎC(Fig.52D + 8)ƕctPol α , 3'½+ Oz *ĺ% dGTP BÂ«*õ=ʎC&= (Fig.52E + 7)dGTP +õ=ʎ6ãǪ;=,à> dATP ' dCTP +õ=ʎ696<?
(Fig.52E + 6 ' 8)5hPol β , dGTP BÂ«*õ=ʎC&

 (Fig.52F + 7)¥Ü+ 1 #+ Oz *ĺ>Ěė+õ=ʎ6ɥƫ&,KF exo-, Oz *ĺ% dGTP
' dATP BctPol αhPol β , dGTP BÂ«*õ=ʎ7'ƛ<
ƨȊȖȱƭ

')"%

<Oz ʔȳʠÖ+ 3'½+ Oz *ĺ>Ěė+õ=ʎ6*

7Ěė+ȓˈ+Àûª%>'ƛ<~]ǍŪ,ȒŎ%9õ=ʎ

')"

KF exo-' Pol αPol β , DNA +ɜɛ:·ş*ʫ> DNA z
?<+ DNA z= 20)

<ˏ~]&= 43)ŧıŎB9"%>ɌǼ*KF

exo-: Pol αPol β *;> DNA ùų,ƈ¿*;"%ŖʿBö:¥Ü+ȊȖȱƭ

<Oz  1 #+ęù, KF exo-: Pol αPol β *;> DNA ùų¼ƻ,)ƛ<' 20)Oz ʔȳʠÖ&,?<+ DNA ùųƈ¿ʟ«&¼ƻ%')"%5"+'<DNA +ɜɛ:·şʘȒ*%Oz +ʔȳʠÖ, DNA z~]*;> DNA ùųB¼ƻ>;=ʣȣ)ƈ¿')>'ȍĄ?

104

Fig.52 DNA synthesis by KF exo-, ctPol α or hPol β across OzOz. (A-C) Primer extension
across OzOz by KF exo- (A), ctPol α (B), or hPol β (C). Decreasing amounts of KF exo- (250µU in lanes 1 and 5, 25 µU in lanes 2 and 6, 2.5 µU in lanes 3 and 7), ctPol α (100 mU in lanes 1
and 5, 33 mU in lanes 2 and 6, 10 mU in lanes 3 and 7) or hPol β (25 mU in lanes 1 and 5, 2.5
mU in lanes 2 and 6, 250 µU in lanes 3 and 7), was incubated with template (containing GG in
lanes 1-4, containing OzOz in lanes 5-8) and 100 µM of each of the four dNTPs. Lanes 4 and 8
contained no enzyme as negative controls. (D-F) Nucleotide selectivity of KF exo- (D), ctPol α(E) or hPol β (F) opposite OzOz. KF exo- (250 µU), ctPol α (100 mU) or hPol β (2.5 mU) was
incubated with template (containing GG in lanes 1-5, or OzOz in lanes 6-10) and 100 µM of asingle dNTP (N = C, G, A, or T) (lanes 1-4 and 6-9). Lanes 5 and 10 contained no enzyme as
negative controls. Reproduced from Suzuki M. et al., J. Biochem., 159, 323-329 (2016) withpermission from the Oxford University Press.

1053-3. hPol ι
hPol ι

;/ hPol κyREV1 + Oz ʔȳʠÖ*ĺ> DNA z;/ hPol κyREV1  Oz ʔȳʠÖB=ʄɥƫȜ 4 ȚŚ,©ʨ)~]óŦɥƫ% DNA ùųBɕ

>

(

B

< hPol ι ' yREV1 , 1 #+ Oz *ĺ%Ěė+6õ=ʎ6+

'ƛ<

')"%

>&hPol ι ' yREV1 , Oz ʔȳʠÖ*ĺ%9Ěė+6õ=ʎ7'ǖ?>8Oz ʔȳʠÖ+ 3'½+ Oz *ĺ>;/ yREV1 ,Uf&>R

Ěė+õ=ʎ6Bɥƫ+ȱƭhPol ι
EiʔȳʠÖ+ 3';/ 5'½+ G *ĺ% dCTP BãǪǼ*õ=ʎC

53B + 3)Oz ʔȳʠÖ*ĺ%, 3'½+ Oz *ĺ%

 (Fig.53A 'ĚėBĿõ=ʎ7+6&" (Fig.53A ' 53B + 8)ƕhPol κ ,Uf&>REiʔȳʠÖBü7 DNA dtfBǰęù&,Éʨ5,Éʨ+ĚėŴÜ

5&©ʨ+*ĺ% (Fig.53C + 1-3)OzOz ʔȳʠÖBü7 DNA dtfBǰęù,ÉĚė+õ=ʎ6ƳÔ?)

hPol ι , Oz ʔȳʠÖ+ 3'½+ Oz *ĺ%A

" (Fig.53C + 5-7)

* dGTP : dATPdTTP Bõ=ʎC(Fig.53A + 9-12)ƕyREV1 , Oz ʔȳʠÖ+ 3'½+ Oz *ĺ%¬ãǪ)< dCTP Bõ=ʎC(Fig.53B + 9)Oz ʔȳʠÖ+ 3'½+ Oz *ĺ>Ěė+õ=ʎ6ãǪ,REiʔȳʠÖ+ęù'ǂʊ>'ʼŋ*¬
53B + 3-6 ' 9-12 Bǂʊ)

Oz ʔȳʠÖ*ĺ%õ=ʎ7Ěė+Àû,Ɋ%43, 96);/ Pol κREV1 ,ƈ¿=ʄ

ęù*ƈ¿ĚėB=ʄ
+ȱƭ+;*Pol ι

>*9

DNA z

% DNA ùųBż~]'%Ȉ<?%=~]*;> DNA ùų¼ƻʣɝ)ŗÝBż"%>Ȝ 4 ȚA<Pol ι%Éʨʏ5& DNA Bùų&>'Bƛ<;/ Pol κREV1 , Oz ʔȳʠÖ+ 3'½5,

5'½+ Oz ʟ«& DNA ùųóŦ¼ƻ'*ʫ> DNA z<hPol ι ' yREV1 ;/ Pol κREV1 , 1 #+ Oz *ĺ%ȒŎ+ãǪ&ĚėBõ=ʎ6Pol κ , 1 #+ Oz B=ʄ*%'

ē+ DNA zƈ¿Ěė*;"% DNA +ɜɛBż;/Ȝ 4 Ț;= hPol ι , Oz *ĺ%É%+ĚėBõ=ʎ6yREV1 , dCTP BÂ«*õ=ʎCPol ι" (Fig.53A~]*ß #<Oz ʔȳʠÖ, DNA +ɜɛ:·ş+ƈ¿=ʄ ē DNA z;> DNA ùų9¼ƻ>ʼŋ*ʣȣ)ƈ¿&>'ȍĄ?
106

~]*

Fig.53 DNA synthesis with hPol ι, hPol κ, or yREV1 across OzOz. (A, B) Primer extension
across OzOz and nucleotide incorporation opposite OzOz by hPol ι (A) or yREV1 (B). hPol ι(0.5 µg) or yREV1 (4.2 ng) was incubated with template (containing GG in lanes 1-6, containing
OzOz in lanes 7-12) and 100 µM of each of the four dNTPs (lanes 2 and 8) or 100 µM of asingle dNTP (N = C, G, A, or T) (lanes 3-6 and 9-12). Lanes 1 and 7 contained no enzyme as
negative controls. (C) Primer extension across OzOz by hPol κ. Decreasing amounts of hPol κ(31 ng in lanes 1 and 5, 9.2 ng in lanes 2 and 6, 3.1 ng in lanes 3 and 7) were incubated with
template (containing GG in lanes 1-4, containing OzOz in lanes 5-8) and 100 µM of each of thefour dNTPs. Lanes 4 and 8 contained no enzyme as negative controls. Reproduced from Suzuki
M. et al., J. Biochem., 159, 323-329 (2016) with permission from the Oxford University Press.

1073-4. hPol η + Oz ʔȳʠÖ*ĺ> DNA z
Pol η 9ƈ¿=ʄɛBɕ

ē+ DNA z~]óŦ
= 43, 96)ɲ=!)ɜ

~]'%Ȉ<?%ǍŪ> 88-90)DNA Bƽ8>;)ƈ¿&> CPD *ʫ%,Ƽȋ

% DNA Bùų&> 88)51 #+ Oz 9=ʄãǪǼ*=ʄ&>'ƛ<')"%% DNA Bùų> 21)+8KF exo-: Pol αPol βPol ιPol κ

REV1 *;> DNA ùųB¼ƻ> Oz ʔȳʠÖ*#%9Pol η ,=ʄɥƫ+ȱƭhPol η  Oz ʔȳʠÖB=ʄ

Bùų&>'Ⱥ#% DNA%Éʨ5& DNABùųmgƳÔ? (Fig.54A + 5-7)+ãǪ,REiʔȳʠÖ: CPD Bǰ" (Fig.54A + 5-7 ' 1-3 5ęù*ǂ2>'ʼŋ*¬, 9-11 Bǂʊ)5hPol η *;> DNA ùų+Ġ, Oz ʔȳʠÖ+ 3'5, 5'½+ Oz ʟ«&¼ƻ%>9+ĠƳÔ? (Fig.54A + 5)

hPol η ,REiʔȳʠÖ+ 3'
dATPdTTP Bõ=ʎC&;/ 5'½+ G *ĺ% dCTP

= (Fig.54B + 1-4)Oz ʔȳʠÖ+ęù&9úƶ*

dCTP ' dGTPdATPdTTP Bõ=ʎC&

 (Fig.54B + 6-9) 5REiʔȳʠÖ+ęù*ǂ2>' Oz ʔȳʠÖ*

>Ěė+õ=ʎ6ãǪ,¬(Fig.54B + 1-4 ' 6-9 Bǂʊ)Ěė+õ=ʎ6ɥƫʠÖ' Oz ʔȳʠÖ+ƕ*)ƽCOz ʔȳʠÖB=ʄ

% DNA Bùų>'

=ʎC

'

'ƛ<

DNA dtfB=ʄ)<Pol η , Oz ʔȳʠÖ+ 3'+ Oz"<hPol η ,REiʔȳ%ɲ=!) DNA ùųBɕƨȊȖȱƭ,CPD +;&)dGTP :')"<?> hPol η &)=čʸ&>'Bȍ&) 5'½+ Oz *ĺ%ĚėBõǞ&,?5&ɥƫ KF exo-: Pol αPol βPol ιPol κREV1',ġǵ)"%+ȱƭ,Oz ʔȳʠÖ*;"% DNA ɜɛ¼ƻ'%9Pol η  Oz ʔȳʠÖ*ĺ%ĚėBõ=ʎ6+Ś¢+ DNA z~]*

ZGcbR>'&DNA +ɜɛÎ/ʩĥ&>÷ȿŪ>'Bȍ

108

Fig.54 DNA synthesis with hPol η across OzOz. (A) Primer extension across OzOz by hPol η.
Decreasing amounts of hPol η (12 ng in lanes 1, 5, and 9, 4.0 ng in lanes 2, 6, and 10, 1.2 ng inlanes 3, 7, and 11) were incubated with template (containing GG in lanes 1-4, containing OzOz
in lanes 5-8, containing CPD in lanes 9-12) and 100 µM of each of the four dNTPs. Lanes 4, 8,
and 12 contained no enzyme as negative controls. (B) Nucleotide selectivity of hPol η opposite
OzOz. hPol η (4.0 ng) was incubated with template (containing GG in lanes 1-5, and OzOz inlanes 6-10) and 100 µM of a single dNTP (N = C, G, A, or T) (lanes 1-4 and 6-9). Lanes 5 and
10 contained no enzyme as the negative control. Reproduced from Suzuki M. et al., J. Biochem.,
159, 323-329 (2016) with permission from the Oxford University Press.

1093-5. yPol ζ + Oz ʔȳʠÖ*ĺ> DNA z
Pol ζ 9ƈ¿=ʄ

ē+ DNA z~]óŦ

~]&> 43)¢+ DNA z

úƶ* Oz ʔȳʠÖ& DNA ùų¼ƻ>+DNA ùųBɕ~]'?'9 Oz ʔȳʠÖB=ʄ

%'&>+Bɥƫˌ2'*yPol ζ ,¢+ DNA z~]',ǵ)=Oz ʔȳʠÖB=ʄ
& (Fig.55A + 5-7)

%Éʨ5& DNA ùųBãǪɍɕ

Uf'%ǰ

+ęù'ǂʊ>'Oz ʔȳʠÖB=ʄ©ʨãǪ,¬

'

REiʔȳʠÖ" (Fig.55A +

5-7 ' 1-3 Bǂʊ)
Uf'%ǰ

REiʔȳʠÖBü7 DNA dtf+ęùPol ζ, dCTP +6ĩđ&9 17-mer 5&tG{B©ʨ (Fig.55B + 1)dGTP :dTTP 9REiʔȳʠÖ+ 3'½+ G *ĺ%õ=ʎC
ƕOz ʔȳʠÖ+ęù,3'(Fig.55B + 2 ' 4);/ 5'½+ Oz *ĺ> dGTP +õ=ʎ6ɟ<?

(Fig.55B + 7)3'½+ Oz *ĺ> dCTP ' dTTP +õ=ʎ696<? (Fig.55B+ 6 ' 9)REiʔȳʠÖ+ęù;=9ǔĿ%/ 9 ' 1

 (Fig.55B + 6;;/ 4 Bǂʊ)ƕOz ʔȳʠÖ*ĺ> dATP +õ=ʎ6,REiʔȳʠÖ+ęù;=9Ĝß% (Fig.55B + 8 ' 3 Bǂʊ)

?<+ȱƭ,Pol ζ , Oz  1 #&9ʔȳʠÖ&9ʫ´)ɲ=!)Ěė+õ=ʎ
6Bɕ

'BȍPol ζ ,ƈ¿'* DNA z~]ǍŪƶŐǵ)>ƈ¿=ʄ~]&=UV *;>ȘǠĞǵɰǻ*%ʣɝ)ŗÝBż"%ē+ DNA z>'Ȉ<> 47, 54, 55)5Ȝ 4 Ț&,Pol ζ , 1 #+ Oz *ĺ%ãǪɍ=ʄ

?%DNA Bùų+ãǪ, G Bü7 DNA dtfBǰùų*ęù'34úȒŎ&

')"%

>¥

<Oz ʔȳʠÖB Pol ζ ãǪɍ=ʄ<?

<Oz  1 #

ʔȳ%

>

*ʫ´)Pol ζ  Oz +ƈ¿=ʄ

DNA>'ƛ<>'%%ʣɝ)ŗÝBż"%

>+&,)

110

'Ⱥ

%

>

Fig.55 DNA synthesis with yPol ζ across OzOz. (A) Primer extension across OzOz by yPol ζ.
Decreasing amounts of yPol ζ (50 ng in lanes 1 and 5, 17 ng in lanes 2 and 6, 5.0 ng in lanes 3
and 7) were incubated with template (containing GG in lanes 1-4, containing OzOz in lanes 5-8)
and 100 µM of each of the four dNTPs. Lanes 4 and 8 contained no enzyme as negative controls.
(B) Nucleotide selectivity of yPol ζ opposite OzOz. yPol ζ (17 ng) was incubated with template(containing GG in lanes 1-5, and OzOz in lanes 6-10) and 100 µM of a single dNTP (N = C, G,
A, or T) (lanes 1-4 and 6-9). Lanes 5 and 10 contained no enzyme as negative controls.
Reproduced from Suzuki M. et al., J. Biochem., 159, 323-329 (2016) with permission from theOxford University Press.

111

Ȝ 4 ȡ ȱɶ
KF exo-: Pol αPol βPol ιREV1 , Oz ʔȳʠÖ+ 3'+ Oz *ĺ%Ěėõ=ʎ
6+ãǪ,REiʔȳʠÖ+ęù;=9¬Oz ʔȳʠÖ+ 5'+ Oz *ĺ>Ě
ė+õ=ʎ6,œʭĴ?>'ƛ<')" (Table 12)5Oz ʔȳʠÖ+3'+ Oz *ĺ%õ=ʎ7Ěė,??+ DNA zƕPol κ , Oz ʔȳʠÖ+ 3'+ Oz &<Oz ʔȳʠÖ,ƈ¿=ʄ

+ DNA zĚė+õ=ʎ6,ɟ<?Oz ʔȳʠÖ,Pol κ *;> DNA ùųBĮÉ*ʭĴ>'ƛ<ƭ~]*;"%ǵ)"%')" (Table 12)?<+ȱDNA ùųBż

DNA z~]9ü8Ġ~]*;> DNA ùųB¼ƻ>ƈ¿&>'ȍ?

CPD BãǪɍ=ʄ

% DNA Bùų&> Pol η &,Oz ʔȳʠÖB=ʄDNA ùų9ƳÔ? (Table 12)+Ġ, Oz ʔȳʠÖ+ 3'5, 5'½+ Oz &¼ƻ%>'ƛ<')"¥<Oz ,ʔȳʠÖ'%ǯų>'Ğǵï

Ū+ˏ;=97@DNA ùųBʭĴ>;)ʼŋ*ʣȣ)ƈ¿')>'ȍĄ

?
?*ĺ%Pol ζ , Oz ʔȳʠÖ*ĺ%ɲ=!)ĚėBõ=ʎ7¢+ DNAz~]',ǵ)=Oz ʔȳʠÖB=ʄ%ãǪɍ DNA Bùų&>'')" (Table 12)ƨȊȖȱƭ,Oz ʔȳʠÖB=ʄƛ<ɍɕ'&> DNA z

 DNA ùųBãǪ

~]'%+ Pol ζ +÷ȿŪB×8%ȍ?*;=Oz ʔȳʠÖǯųęù*¢+ DNA z~]*;> DNA ùų

Oz ʔȳʠÖʟ«&¼ƻ'%91 Pol ζ Qf?>' DNA ùų+¼ƻBċʞ&>'ȍĄ?+8Ś, Oz ʔȳʠÖ*ʫ>ƈ¿=

DNA ùų+ɮȭ)~Mi[}+ɥƫŤɝ&>'Ⱥ

ʄ

¥*ß

<?>

Oz ʔȳʠÖ*ĺ%õ=ʎ5?>Ěė*ȅȀęùOz *ĺ>dGTP +õ=ʎ6 REV1 ' KF exo-BʰÉ%+ DNA z
?5&+ȊȖĘþ 20, 21);/Ȝ 4 Ț*%9REV1 BʰÉ%+ DNA z~]*Ëʑ%

> Oz ë­*ĺ>Ěė+õ=ʎ6*

~]*

112

% Oz *ĺ> dGTP +õ=ʎ6Ëʑ%'9Ⱥű>'Oz ,Ǧ*Ȅưǯǥ*Y+ǻǯ*ʫ>+&,)

'œȺ

%

% Oz  G:C-C:G fZm>Table 12 Summary of chapter 6.
Insertion

Extension

opposite 3' Oz

opposite 5' Oz

Replicative DNA polymerases

Pol αG, A, Cstall

DNA polymerases

KF exo-

A



stall

involved in DNA repair

Pol β

G



stall

Pol ζ

G, A, C, T

G, A

high efficiency

DNA polymerases

Pol ηG, A, C, T

G, A, C, T

low efficiency

involved in translesion

Pol ιG, A, T

stallsynthesis

Pol κ





stall

REV1

C



stall

113

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(1)

Steenken, S., and Jovanovic, S. V. (1997) How easily oxidizable is DNA? One-electron
reduction potentials of adenosine and guanosine radicals in aqueous solution. J. Am.
Chem. Soc., 119, 617-618.

(2)Kino, K., and Sugiyama, H. (2005) UVR-induced G-C to C-G transversions fromoxidative DNA damage. Mutat. Res., 571, 33-42.

(3)Khaled, H. M., Bahnassi, A. A., Zekri, A. R., Kassem, H. A., and Mokhtar, N. (2003)
Correlation between p53 mutations and HPV in bilharzial bladder cancer. Urol. Oncol.,21, 334-341.

(4)

Maehira, F., Miyagi, I., Asato, T., Eguchi, Y., Takei, H., Nakatsuki, K., Fukuoka, M.,
and Zaha, F. (1999) Alterations of protein kinase C, 8-hydroxydeoxyguanosine, andK-ras oncogene in rat lungs exposed to passive smoking. Clin. Chim. Acta, 289, 133-144.

(5)

Giaretti, W., Rapallo, A., Geido, E., Sciutto, A., Merlo, F., Risio, M., and Rossini, F. P.
(1998) Specific K-ras2 mutations in human sporadic colorectal adenomas are associatedwith DNA near-diploid aneuploidy and inhibition of proliferation. Am. J. Pathol., 153,
1201-1209.

(6)

Sugio, K. (2009) Ras mutation ƳÔǈ+meX+ǫǧ. #-403,

7, 195-202.
(7)

Suzuki, M., Kino, K., and Miyazawa, H. (2012) REiʢçƈ¿*ĺ> DNA z~]+ĚėʜŹŪ. Rad. Biol. Res. Comm., 47, 137-164.

(8)

Oda, Y., Uesugi, S., Ikehara, M., Nishimura, S., Kawase, Y., Ishikawa, H., Inoue, H., andOhtsuka, E. (1991) NMR studies of a DNA containing 8-hydroxydeoxyguanosine.
Nucleic Acids Res., 19, 1407-1412.

(9)

McAuley-Hecht, K. E., Leonard, G. A., Gibson, N. J., Thomson, J. B., Watson, W. P.,
Hunter, W. N., and Brown, T. (1994) Crystal structure of a DNA duplex containing
8-hydroxydeoxyguanine-adenine base pairs. Biochemistry, 33, 10266-10270.

(10)

Lipscomb, L. A., Peek, M. E., Morningstar, M. L., Verghis, S. M., Miller, E. M., Rich, A.,118

Essigmann, J. M., and Williams, L. D. (1995) X-ray structure of a DNA decamer
containing 7,8-dihydro-8-oxoguanine. Proc. Natl. Acad. Sci. U.S.A., 92, 719-723.
(11)

Cadet, J., Berger, M., Buchko, G. W., Joshi, P. C., Raoul, S., and Ravanat, J.-L. (1994)
2,2-Diamino-4-[(3,5-di-O-acetyl-2-deoxy-β-D-erythro-pentofuranosyl)amino]-5-(2H)-ox
azolone:

A

novel

and

predominantradicaloxidation

productof

3',5'-di-O-acetyl-2'-deoxyguanosine. J. Am. Chem. Soc., 116, 7403-7404.
(12)

Luo, W., Muller, J. G., and Burrows, C. J. (2001) The pH-dependent role of superoxide
in riboflavin-catalyzed photooxidation of 8-oxo-7,8-dihydroguanosine. Org. Lett., 3,2801-2804.

(13)

Kino, K., Saito, I., and Sugiyama, H. (1998) Product analysis of GG-specificphotooxidation of DNA via electron transfer:# 2-aminoimidazolone as a major guanineoxidation product. J. Am. Chem. Soc., 120, 7373-7374.

(14)

Morikawa, M., Kino, K., Oyoshi, T., Suzuki, M., Kobayashi, T., and Miyazawa, H.
(2013)

Product

analysisofphotooxidationinisolated

quadruplex

DNA;

8-oxo-7,8-dihydroguanine and its oxidation product at 3'-G are formed instead of
2,5-diamino-4H-imidazol-4-one. RSC Adv., 3, 25694.
(15)

Morikawa, M., Kino, K., Oyoshi, T., Suzuki, M., Kobayashi, T., and Miyazawa, H.
(2014) Analysis of guanine oxidation products in double-stranded DNA and proposed
guanine oxidation pathways in single-stranded, double-stranded or quadruplex DNA.
Biomolecules, 4, 140-159.

(16)

Kino, K., and Sugiyama, H. (2001) Possible cause of G-C-->C-G transversion mutation
by guanine oxidation product, imidazolone. Chem. Biol., 8, 369-378.

(17)

Neeley, W. L., Delaney, J. C., Henderson, P. T., and Essigmann, J. M. (2004) In vivo
bypass efficiencies and mutational signatures of the guanine oxidation products
2-aminoimidazolone

and5-guanidino-4-nitroimidazole.

J.

Biol.

Chem.,

279,

43568-43573.
(18)

Matter, B., Malejka-Giganti, D., Csallany, A. S., and Tretyakova, N. (2006) Quantitative
analysisoftheoxidativeDNAlesion,119

2,2-diamino-4-(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope
dilution-capillary HPLC-ESI-MS/MS. Nucleic Acids Res, 34, 5449-5460.
(19)

Ming, X., Matter, B., Song, M., Veliath, E., Shanley, R., Jones, R., and Tretyakova, N.
(2014) Mapping structurally defined guanine oxidation products along DNA duplexes:
influence of local sequence context and endogenous cytosine methylation. J. Am. Chem.
Soc., 136, 4223-4235.

(20)

Kino, K., Sugasawa, K., Mizuno, T., Bando, T., Sugiyama, H., Akita, M., Miyazawa, H.,
and Hanaoka, F. (2009) Eukaryotic DNA polymerases α, β and ε incorporate guanine
opposite 2,2,4-triamino-5(2H)-oxazolone. ChemBioChem, 10, 2613-2616.

(21)

Kino, K., Ito, N., Sugasawa, K., Sugiyama, H., and Hanaoka, F. (2004) Translesion
synthesis by human DNA polymerase η across oxidative products of guanine. Nucleic
Acids Symp. Ser., 171-172.

(22)

Kino, K., Morikawa, M., Kobayashi, T., Kobayashi, T., Komori, R., Sei, Y., andMiyazawa, H. (2010) The oxidation of 8-oxo-7,8-dihydroguanine by iodine. Bioorg. Med.
Chem. Lett., 20, 3818-3820.

(23)

Luo, W., Muller, J. G., Rachlin, E. M., and Burrows, C. J. (2000) Characterization of
spiroiminodihydantoin

asaproductof

one-electronoxidationof

8-oxo-7,8-dihydroguanosine. Org. Lett., 2, 613-616.
(24)

Ye, Y., Munk, B. H., Muller, J. G., Cogbill, A., Burrows, C. J., and Schlegel, H. B.
(2009)

Mechanistic

aspects

oftheformationofguanidinohydantoin

fromspiroiminodihydantoin under acidic conditions. Chem. Res. Toxicol., 22, 526-535.
(25)

Luo, W., Muller, J. G., Rachlin, E. M., and Burrows, C. J. (2001) Characterization of
hydantoin products from one-electron oxidation of 8-oxo-7,8-dihydroguanosine in a
nucleoside model. Chem. Res. Toxicol., 14, 927-938.

(26)

Kornyushyna, O., Berges, A. M., Muller, J. G., and Burrows, C. J. (2002) In vitro
nucleotide misinsertion opposite the oxidized guanosine lesions spiroiminodihydantoin
and guanidinohydantoin and DNA synthesis past the lesions using Escherichia coli DNA
polymerase I (Klenow Fragment). Biochemistry, 41, 15304-15314.
120

(27)

Shibutani, S., Takeshita, M., and Grollman, A. P. (1997) Translesional synthesis on DNA
templates containing a single abasic site. A mechanistic study of the “A rule”. J. Biol.
Chem., 272, 13916-13922.

(28)

Matray, T. J., and Kool, E. T. (1999) A specific partner for abasic damage in DNA.
Nature, 399, 704-708.

(29)

Beckman, J., Wang, M., Blaha, G., Wang, J., and Konigsberg, W. H. (2010) Substitutionof Ala for Tyr567 in RB69 DNA polymerase allows dAMP and dGMP to be inserted
opposite Guanidinohydantoin. Biochemistry, 49, 8554-8563.

(30)

Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J.
R., Montgomery, J. A., Vreven, T. J., Kudin, K. N., and Burant, J. C. et al. Gaussian 03,
Revision C.02; Gaussian, Inc.: Wallingford, CT, USA, 2004.

(31)

Ding, S., Jia, L., Durandin, A., Crean, C., Kolbanovskiy, A., Shafirovich, V., Broyde, S.,
and Geacintov, N. E. (2009) Absolute configurations of spiroiminodihydantoin andallantoin stereoisomers: comparison of computed and measured electronic circulardichroism spectra. Chem. Res. Toxicol., 22, 1189-1193.

(32)

Wong, M. W., Frisch, M. J., and Wiberg, K. B. (1991) Solvent effects. 1. The mediationof electrostatic effects by solvents. J. Am. Chem. Soc., 113, 4776-4782.

(33)

Verdolino, V., Cammi, R., Munk, B. H., and Schlegel, H. B. (2008) Calculation of pKa
values of nucleobases and the guanine oxidation products guanidinohydantoin andspiroiminodihydantoin using density functional theory and a polarizable continuum
model. J. Phys. Chem. B, 112, 16860-16873.

(34)

Aller, P., Ye, Y., Wallace, S. S., Burrows, C. J., and Doublie, S. (2010) Crystal structureofareplicativeDNA

polymerase

bound

to

the

oxidized

guaninelesionguanidinohydantoin. Biochemistry, 49, 2502-2509.
(35)

Chinyengetere, F., and Jamieson, E. R. (2008) Impact of the oxidized guanine lesion
spiroiminodihydantoin on the conformation and thermodynamic stability of a 15-mer
DNA duplex. Biochemistry, 47, 2584-2591.

(36)

Yanson, I. K., Teplitsky, A. B., and Sukhodub, L. F. (1979) Experimental studies of121molecular interactions between nitrogen bases of nucleic acids. Biopolymers, 18,1149-1170.
(37)

Prakash, S., and Prakash, L. (2002) Translesion DNA synthesis in eukaryotes: a one- or
two-polymerase affair. Genes Dev., 16, 1872-1883.

(38)

Morikawa, M., Kino, K., and Miyazawa, H. (2010) DNA ƈ¿+ǻǯ'ȭȾ1+Ŗʿ*ʫ>çĪǼɠǞ. Rad. Biol. Res. Comm., 45, 268-285.

(39)

Wu, M., Yan, S., Patel, D. J., Geacintov, N. E., and Broyde, S. (2002) Relating repair
susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic
stability. Nucleic Acids Res., 30, 3422-3432.

(40)

Sawaya, M. R., Prasad, R., Wilson, S. H., Kraut, J., and Pelletier, H. (1997) Crystal
structures of human DNA polymerase β complexed with gapped and nicked DNA:evidence for an induced fit mechanism. Biochemistry, 36, 11205-11215.

(41)

Neeley, W. L., Delaney, S., Alekseyev, Y. O., Jarosz, D. F., Delaney, J. C., Walker, G. C.,
and Essigmann, J. M. (2007) DNA polymerase V allows bypass of toxic guanineoxidation products in vivo. J. Biol. Chem., 282, 12741-12748.

(42)

Garg, P., and Burgers, P. M. (2005) DNA polymerases that propagate the eukaryotic
DNA replication fork. Crit. Rev. Biochem. Mol. Biol., 40, 115-128.

(43)

Pavlov, Y. I., Shcherbakova, P. V., and Rogozin, I. B. (2006) Roles of DNA polymerases
in replication, repair, and recombination in eukaryotes. Int. Rev. Cytol., 255, 41-132.

(44)

Rothwell, P. J., and Waksman, G. (2005) Structure and mechanism of DNA polymerases.
Adv. Protein Chem., 71, 401-440.

(45)

Nick McElhinny, S. A., Gordenin, D. A., Stith, C. M., Burgers, P. M., and Kunkel, T. A.
(2008) Division of labor at the eukaryotic replication fork. Mol. Cell, 30, 137-144.

(46)

Pursell, Z. F., Isoz, I., Lundstrom, E. B., Johansson, E., and Kunkel, T. A. (2007) Yeast
DNA polymerase ε participates in leading-strand DNA replication. Science, 317,
127-130.

(47)

Zhu, F., and Zhang, M. (2003) DNA polymerase ζ: new insight into eukaryotic
mutagenesis and mammalian embryonic development. World J. Gastroenterol., 9,1221165-1169.
(48)

Yang, W., and Woodgate, R. (2007) What a difference a decade makes: insights intotranslesion DNA synthesis. Proc. Natl. Acad. Sci. U.S.A., 104, 15591-15598.

(49)

Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D.,
Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo,
T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) The Y-family of DNApolymerases. Mol. Cell, 8, 7-8.

(50)

Fazlieva, R., Spittle, C. S., Morrissey, D., Hayashi, H., Yan, H., and Matsumoto, Y.
(2009) Proofreading exonuclease activity of human DNA polymerase δ and its effects on
lesion-bypass DNA synthesis. Nucleic Acids Res., 37, 2854-2866.

(51)

Piao, J., Masuda, Y., and Kamiya, K. (2010) Specific amino acid residues are involved in
substrate discrimination and template binding of human REV1 protein. Biochem. Biophys.
Res. Commun, 392, 140-144.

(52)

MacNeill, S. A., Baldacci, G., Burgers, P. M., and Hubscher, U. (2001) A unified
nomenclature for the subunits of eukaryotic DNA polymerase δ. Trends Biochem. Sci.,
26, 16-17.

(53)

Jin, Y. H., Garg, P., Stith, C. M., Al-Refai, H., Sterling, J. F., Murray, L. J., Kunkel, T.
A., Resnick, M. A., Burgers, P. M., and Gordenin, D. A. (2005) The multiple biologicalroles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase δ requireswitching between the polymerase and exonuclease domains. Mol. Cell. Biol., 25,
461-471.

(54)

Lawrence, C. W. (2002) Cellular roles of DNA polymerase ζ and Rev1 protein. DNA
Repair (Amst), 1, 425-435.

(55)

Lawrence, C. W. (2004) Cellular functions of DNA polymerase ζ and Rev1 protein. Adv.
Protein Chem., 69, 167-203.

(56)

Guo, D., Wu, X., Rajpal, D. K., Taylor, J. S., and Wang, Z. (2001) Translesion synthesisby yeast DNA polymerase ζ from templates containing lesions of ultraviolet radiation
and acetylaminofluorene. Nucleic Acids Res., 29, 2875-2883.
123

(57)

Haracska, L., Prakash, S., and Prakash, L. (2003) Yeast DNA polymerase ζ is an efficient
extender of primer ends opposite from 7,8-dihydro-8-oxoguanine and O6-methylguanine.
Mol. Cell. Biol., 23, 1453-1459.

(58)

Haracska, L., Unk, I., Johnson, R. E., Johansson, E., Burgers, P. M., Prakash, S., andPrakash, L. (2001) Roles of yeast DNA polymerases δ and ζ and of Rev1 in the bypass ofabasic sites. Genes Dev., 15, 945-954.

(59)

Gan, G. N., Wittschieben, J. P., Wittschieben, B. O., and Wood, R. D. (2008) DNA
polymerase zeta (pol ζ) in higher eukaryotes. Cell Res., 18, 174-183.

(60)

Andersen, P. L., Xu, F., Ziola, B., McGregor, W. G., and Xiao, W. (2011) Sequential
assembly of translesion DNA polymerases at UV-induced DNA damage sites. Mol. Biol.
Cell, 22, 2373-2383.

(61)

Xie, W., Yang, X., Xu, M., and Jiang, T. (2012) Structural insights into the assembly ofhuman translesion polymerase complexes. Protein Cell, 3, 864-874.

(62)

Tissier, A., Frank, E. G., McDonald, J. P., Iwai, S., Hanaoka, F., and Woodgate, R.
(2000) Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase ι.
EMBO J., 19, 5259-5266.

(63)

Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000)
Eukaryotic polymerases ι and ζ act sequentially to bypass DNA lesions. Nature, 406,
1015-1019.

(64)

Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Preferential incorporation of G
opposite template T by the low-fidelity human DNA polymerase ι. Mol. Cell Biol., 20,
7099-7108.

(65)

Choi, J. Y., and Guengerich, F. P. (2006) Kinetic evidence for inefficient and error-prone
bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι. J. Biol.
Chem., 281, 12315-12324.

(66)

Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) polι, a remarkably
error-prone human DNA polymerase. Genes Dev., 14, 1642-1650.

(67)

McDonald, J. P., Tissier, A., Frank, E. G., Iwai, S., Hanaoka, F., and Woodgate, R.
124

(2001) DNA polymerase iota and related rad30-like enzymes. Philos. Trans. R. Soc.
Lond Ser. B, 356, 53-60.
(68)

Zhang, Y., Yuan, F., Wu, X., Taylor, J. S., and Wang, Z. (2001) Response of human
DNA polymerase ι to DNA lesions. Nucleic Acids Res., 29, 928-935.

(69)

Zhang, Y., Yuan, F., Wu, X., Wang, M., Rechkoblit, O., Taylor, J. S., Geacintov, N. E.,
and Wang, Z. (2000) Error-free and error-prone lesion bypass by human DNA
polymerase κ in vitro. Nucleic Acids Res., 28, 4138-4146.

(70)

Zhang, Y., Yuan, F., Xin, H., Wu, X., Rajpal, D. K., Yang, D., and Wang, Z. (2000)
Human DNA polymerase κ synthesizes DNA with extraordinarily low fidelity. Nucleic
Acids Res., 28, 4147-4156.

(71)

Choi, J. Y., Angel, K. C., and Guengerich, F. P. (2006) Translesion synthesis acrossbulky N2-alkyl guanine DNA adducts by human DNA polymerase κ. J. Biol. Chem., 281,
21062-21072.

(72)

Wolfle, W. T., Washington, M. T., Prakash, L., and Prakash, S. (2003) Human DNA
polymerase κ uses template-primer misalignment as a novel means for extending
mispaired termini and for generating single-base deletions. Genes Dev., 17, 2191-2199.

(73)

Nelson, J. R., Lawrence, C. W., and Hinkle, D. C. (1996) Deoxycytidyl transferase
activity of yeast REV1 protein. Nature, 382, 729-731.

(74)

Zhang, Y., Wu, X., Rechkoblit, O., Geacintov, N. E., Taylor, J. S., and Wang, Z. (2002)
Response of human REV1 to different DNA damage: preferential dCMP insertion
opposite the lesion. Nucleic Acids Res., 30, 1630-1638.

(75)

Masuda, Y., and Kamiya, K. (2002) Biochemical properties of the human REV1 protein.
FEBS Lett., 520, 88-92.

(76)

Choi, J. Y., and Guengerich, F. P. (2008) Kinetic analysis of translesion synthesis
opposite bulky N2- and O6-alkylguanine DNA adducts by human DNA polymerase
REV1. J. Biol. Chem., 283, 23645-23655.

(77)

Swan, M. K., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2009)
Structure of the human Rev1-DNA-dNTP ternary complex. J. Mol. Biol., 390, 699-709.
125

(78)

Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S., and Aggarwal, A. K. (2005) Rev1
employs a novel mechanism of DNA synthesis using a protein template. Science, 309,
2219-2222.

(79)

Howell, C. A., Prakash, S., and Washington, M. T. (2007) Pre-steady-state kinetic studies
of protein-template-directed nucleotide incorporation by the yeast Rev1 protein.
Biochemistry, 46, 13451-13459.

(80)

Kino, K., Takao, M., Miyazawa, H., and Hanaoka, F. (2012) A DNA oligomer containing
2,2,4-triamino-5(2H)-oxazolone is incised by human NEIL1 and NTH1. Mutat. Res., 734,
73-77.

(81)

Murakumo, Y., Ogura, Y., Ishii, H., Numata, S., Ichihara, M., Croce, C. M., Fishel, R.,
and Takahashi, M. (2001) Interactions in the error-prone postreplication repair proteins
hREV1, hREV3, and hREV7. J. Biol. Chem., 276, 35644-35651.

(82)

Ohashi, E., Murakumo, Y., Kanjo, N., Akagi, J., Masutani, C., Hanaoka, F., and Ohmori,
H. (2004) Interaction of hREV1 with three human Y-family DNA polymerases. Genes
Cells, 9, 523-531.

(83)

Kim, M. S., Machida, Y., Vashisht, A. A., Wohlschlegel, J. A., Pang, Y. P., and Machida,
Y. J. (2013) Regulation of error-prone translesion synthesis by Spartan/C1orf124.
Nucleic Acids Res., 41, 1661-1668.

(84)

Acharya, N., Johnson, R. E., Pages, V., Prakash, L., and Prakash, S. (2009) Yeast Rev1
protein promotes complex formation of DNA polymerase ζ with Pol32 subunit of DNApolymerase δ. Proc. Natl. Acad. Sci. U.S.A., 106, 9631-9636.

(85)

Kikuchi, S., Hara, K., Shimizu, T., Sato, M., and Hashimoto, H. (2012) Structural basisof recruitment of DNA polymerase ζ by interaction between REV1 and REV7 proteins. J.
Biol. Chem., 287, 33847-33852.

(86)

Yang, L., Beard, W., Wilson, S., Roux, B., Broyde, S., and Schlick, T. (2002) Local
deformations revealed by dynamics simulations of DNA polymerase β with DNA
mismatches at the primer terminus. J. Mol. Biol., 321, 459-478.

(87)

Beard, W. A., Shock, D. D., and Wilson, S. H. (2004) Influence of DNA structure on
126

DNA polymerase β active site function: extension of mutagenic DNA intermediates. J.
Biol. Chem., 279, 31921-31929.
(88)

Masutani, C., Kusumoto, R., Iwai, S., and Hanaoka, F. (2000) Mechanisms of accuratetranslesion synthesis by human DNA polymerase η. EMBO J., 19, 3100-3109.

(89)

Matsuda, T., Bebenek, K., Masutani, C., Rogozin, I. B., Hanaoka, F., and Kunkel, T. A.
(2001) Error rate and specificity of human and murine DNA polymerase η. J. Mol. Biol.,
312, 335-346.

(90)

Kano, C., Hanaoka, F., and Wang, J. Y. (2012) Analysis of mice deficient in both REV1
catalytic activity and POLH reveals an unexpected role for POLH in the generation of C
to G and G to C transversions during Ig gene hypermutation. Int. Immunol., 24, 169-174.

(91)

Sugiyama, H., and Saito, I. (1996) T Theoretical studies of GG-specific photocleavage of
DNA via electron transfer: significant lowering of ionization potential and 5’-localizationof HOMO of stacked GG bases in B-form DNA. J. Am. Chem. Soc., 118, 7063-7068.

(92)

Saito, I., Nakamura, T., Nakatani, K., Yoshioka, Y., Yamaguchi, K., and Sugiyama, H.
(1998) Mapping of the hot spots for DNA damage by one-electron oxidation: efficacy of
GG doublets and GGG triplets as a trap in long-range hole migration. J. Am. Chem. Soc.,
120, 12686-12687.

(93)

Hirakawa, K., Kawanishi, S., Hirano, T., and Segawa, H. (2007) Guanine-specific DNA
oxidation photosensitized by the tetraphenylporphyrin phosphorus(V) complex via
singlet oxygen generation and electron transfer. J. Photochem. Photobiol. B, 87, 209-217.

(94)

Jaloszynski, P., Masutani, C., Hanaoka, F., Perez, A. B., and Nishimura, S. (2003)
8-Hydroxyguanine in a mutational hotspot of the c-Ha-ras gene causes misreplication,‘action-at-a-distance’ mutagenesis and inhibition of replication. Nucleic Acids Res., 31,6085-6095.

(95)

Jaloszynski, P., Ohashi, E., Ohmori, H., and Nishimura, S. (2005) Error-prone andinefficient replication across 8-hydroxyguanine (8-oxoguanine) in human and mouse ras
gene fragments by DNA polymerase κ. Genes Cells, 10, 543-550.

(96)

Washington, M. T., Carlson, K. D., Freudenthal, B. D., and Pryor, J. M. (2010)127

Variations on a theme: eukaryotic Y-family DNA polymerases. Biochim. Biophys. Acta,
1804, 1113-1123.
(97)

Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M.,
Iwai, S., Takio, K., and Hanaoka, F. (1999) The XPV (xeroderma pigmentosum variant)gene encodes human DNA polymerase η. Nature, 399, 700-704.

(98)

Xiao, W., Lechler, T., Chow, B. L., Fontanie, T., Agustus, M., Carter, K. C., and Wei, Y.
F. (1998) Identification, chromosomal mapping and tissue-specific expression of hREV3
encoding a putative human DNA polymerase ζ. Carcinogenesis, 19, 945-949.

(99)Lin, W., Xin, H., Zhang, Y., Wu, X., Yuan, F., and Wang, Z. (1999) The human REV1
gene codes for a DNA template-dependent dCMP transferase. Nucleic Acids Res., 27,4468-4475.

128

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