鈴木雅代 - 徳島文理大学薬学部

DNA
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DNA
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3
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4
Oz
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1
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5
Oz
DNA
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6
7
Oz
DNA
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DNA
DNA
G:C-T:A
G:C-C:G
p53
K-ras
K-ras
codon 12
G:C-C:G
G:C-T:A
8
(8-oxoG)
8-oxoG
G:C-T:A
8-oxoG
G:C-C:G
8-oxoG
(Iz)
(Oz)
DNA
Oz
(Pol) α
Klenow fragment exonuclease- (KF exo-)
IV
Pol β Pol γ Pol ε Pol η Pol
Oz
G:C-C:G
Oz
G:C-C:G
8-oxoG
(Gh)
(Sp)
(Ia)
KF exoGh/Ia
Sp
Oz
1
Oz
Gh/Ia
Oz
Gh/Ia
Sp
G:C-C:G
G:C-C:G
Oz
2
3
Oz
Gh/Ia Sp
DNA
DNA
DNA
Oz > Gh/Ia > Sp
2
Oz:G
3
Ia:G
sp3
Gh/Ia
DNA
Sp
Sp:G
Oz
sp3
DNA
Gh/Ia
Sp
DNA
Oz
DNA
DNA
DNA
DNA
DNA
Oz
G:C-C:G
Oz
DNA
Pol β Pol γ Pol ε Pol η Pol IV KF exo-
Pol α
Oz
Oz
DNA
4
Oz
DNA
DNA
DNA
Pol ι
DNA
Pol κ REV1
Oz
Pol δ
Oz
5
Oz
REV1
DNA
DNA
DNA
Oz:G
Oz:G
2
Oz:A Oz:C Oz:T
Pol ζ
DNA
KF exo-
6
Pol α Pol β Pol ζ
Pol η
Pol ι
Pol κ REV1
Oz
DNA
4
6
Oz
Oz
REV1
Oz
REV1
DNA
KF exo5
DNA
Oz
DNA
Oz
G:C-C:G
Oz
DNA
REV1
DNA
Oz
Oz
DNA
Pol ζ
Oz
DNA
DNA
Oz
DNA
DNA
Oz
Oz
Pol ζ
DNA
REV1
REV1
Oz
DNA
Oz
Oz
G:C-C:G
Oz
Oz
in vitro
Oz
3
8-oxoG
8-oxo-7,8-dihydroguanine (8
)
2-ME
2-mercaptoethanol
BRCT
BRCA1 C-terminal
BSA
bovine serum albumin
CPD
cyclobutane pyrimidine dimer (
ct
calf thymus
DFT
density functional theory (
Gh
guanidinohydantoin (
h
human
Ia
iminoallantoin (
Iz
2,5-diamino-4H-imidazol-4-one (
KF exo-
Klenow fragment exonuclease-
Oz
2,2,4-triamino-5(2H)-oxazolone (
PMSF
phenylmethylsulfonyl fluoride
Pol
DNA polymerase (DNA
Pur
purine (
Pyr
pyrimidine (
SCRF
self-consistent reaction field (
Sp
spiroiminodihydantoin (
THF
tetrahydrofuran (
Tm
melting temperature (
XP
xeroderma pigmentosum (
y
yeast
)
)
)
)
)
)
)
)
)
)
)
)
)
)
4
1
DNA
DNA
DNA
DNA
DNA
DNA
1)
G:C-T:A
G:C-C:G
(Fig.1)
B2 (
)
K3 (
)
γ
Fe2+
2)
G:C-C:G
3)
p53
K-ras
1)
3-6)
12 13
K-ras
(Table
GTPase
5
CpG
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 type
codon 12
codon 13
colon 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
(8-oxo-7,8-dihydroguanine 8-oxoG)
(Scheme 1)
8-oxoG
7)
8-oxoG:A
(Fig.2) 8-10)
8-oxoG
8-oxoG:C
G:C-T:A
8-oxoG
K-ras
G:C-C:G
(Table 1) 3-6)
8-oxoG
G:C-C:G
O
N
NH
N
dR
N
N
dR
NH 2
N
NH 2
HN
O
N
O
H
N
O
N
dR
Iz
O
∗
N
dR O
Sp
NH 2
NH
N
H
NH 2
∗
N
8-oxoG
NH 2
HN
dR
O
HN
NH
O
G
N
O
H
N
NH 2
O
O
N
∗
N
dR
N
H
Ia
Gh
+H2O
H 2N
N
HN
dR
NH 2
NH
O
O
Oz (closed-ring)
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
H
H N
N 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).
8-oxoG
(Scheme 1) 11-13) Iz
(2,5-diamino-4H-imidazol-4-one Iz)
13-15)
DNA
in vitro
in vivo
Iz
G:C-C:G
16, 17)
Iz
pH 7 37 ºC
147
(Scheme 1) 11)
(2,2,4-triamino-5(2H)-oxazolone Oz)
107
DNA
18)
2 6
Oz
5-
CpG
p53
19)
Oz
Oz
Iz
Oz
DNA
(DNA
Pol β Pol γ Pol ε Pol η Pol IV Klenow fragment exonuclease-
polymerase Pol) α
(KF exo-)
20, 21)
Oz
Oz
G:C-C:G
G:C-C:G
(guanidinohydantoin
(spiroiminodihydantoin Sp)
Sp
Gh)
(Scheme 1) Gh
12, 22-24)
8-oxoG
Gh
(iminoallantoin
(Scheme 1) 25) DNA
8
Ia)
KF exoSp
26)
Gh/Ia
Oz
Oz
20)
Gh/Ia
Gh/Ia
Sp
2
3
G:C-C:G
Oz
G:C-C:G
Oz
Oz
Gh/Ia Sp
DNA
20, 26)
DNA
Pol β Pol γ Pol ε Pol η Pol IV KF exo-
Pol α
Oz
DNA
DNA
DNA
5
Oz
DNA
DNA
9
15
DNA
4
DNA
DNA
6
Oz
Oz
2
(
Molecules, 17, 6705-6715 (2012)
)
1
1
G:C-T:A
G:C-C:G
8-oxoG
G:C-C:G
G:C-C:G
Oz
(Scheme 1)
Gh/Ia
Gh/Ia Sp
Sp
Oz
20, 26)
DNA
DNA
DNA
N-
DNA
27)
A-rule
(tetrahydrofuran
THF)
(Fig.3) 28) THF
G:C
A:T
(
THF
20)
10
)
OH
HO
O
O
OH
HO
Pyrene : THF
Fig.3 The structures of Pyrene:THF base pair 28).
Oz:G
Ia:G
Gh:G
Sp:G
DNA
Oz > Gh/Ia >
20, 26)
Sp
Oz:G
Ia:G
20)
2
Sp:G
2)
Gh:G
29)
Beckman
(density functional theory
SCRF)
Oz:G
Ia:G
DFT)
(self-consistent reaction field
Sp:G
Gh:G
11
Oz:G
2
2'C1'
1
C1'
C1'
2
Gaussian 03 30)
NB3LYP/6-31G**
Onsager reaction field
(ε = 78.39)
(1)
ΔE = E(base pair complex ‘X:Y’)-[E(isolated base ‘X’)+E(isolated base ‘Y’)]
GaussView
Fig.4, 6, 8, 9
12
(1)
3
3-1. Ia:G
Ia
3
kino
2)
3
8
Ia
B3LYP/6-31G**
(Ia1-Ia8)
Ia1:G-Ia8:G
Fig.4
Ia:G
Table 2
Ia1:G
29.5 kcal/mol
C:G
(30.9 kcal/mol)
Ia:G
C:G
3
Ia3:G
Ia4:G Ia5:G Ia7:G Ia8:G
Ia2:G
Ia6:G
29.5 kcal/mol
(O4-H8) (Fig.4B)
28.7 kcal/mol
O4
Ia:G
8
Ia:G
SCRF
Ia1:G
Ia5:G
S
24.1 kcal/mol
Ia1
R
(+)-S
Ia5
Ia5:G
Ia3:G
Ia4:G Ia7:G Ia8:G
Onsager reaction field
13
24.0 kcal/mol
( )-R
31)
Ia1:G
Ia1:G
Ia5:G
Ia1
Ia5
32)
S
Ia3 > Ia4 > Ia1
Ia7 > Ia8 > Ia5
Table
2
Ia1:G
Stabilization
energies
(kcal/mol)
R
Ia5:G
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.
Δ EDFT
Δ ESCRF
Δ 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
Base pair
a
Base pair
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
dR
N6
dR
O
H N
H
7
5
O
N
2
3
4
8
N H
H
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 the
same 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
Beckman
Gh:G
29)
2
33)
(Fig.5A)
1
16
Gh
(Fig.5B) Beckman
(Gh1-Gh16)
3
Gh:G
Gh1:G-Gh16:G
(Fig.6 Table 2) Gh1-Gh16
Gh1-Gh8
( )-S
Gh9-Gh16
Gh:G
(+)-R
2 kcal/mol
Gh3:G
21.4 kcal/mol
16.8 kcal/mol
S
Gh6:G
S
3-1
Gh:G
Gh3:G
Gh11:G
20.6 kcal/mol
Gh3
R
Gh11
S
R
S
R
S
R
12, 34)
Aller
DNA
34)
Gh
S
Aller
Gh:G
Ia:G
Ia:G
Gh/Ia
Gh
Ia
16
A
O
N
HO
∗
N
dR
N
H
H
N
NH
O
O
∗
N
dR
NH 2
enol-1
N
H
OH
N
NH
O
∗
N
dR
NH 2
NH
N
H
NH 2
enol-2
diketo
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
Sp
Ia:G
3
Sp:G
2)
Sp
33)
Sp1
( )-S
Sp2
Sp
2
Sp1
(+)-R
Sp1:G
28.2 kcal/mol
kcal/mol
(Fig.7)
Sp1:G
Sp2:G
18.8 kcal/mol
Sp2:G
(Fig.8 Table 2) 3-1
Sp2:G
Sp:G
Ia1:G
Sp
Ia:G
Kornyushyna
Gh/Ia
Sp
19.9
Sp2:G
Sp1:G
Gh/Ia
Sp2
26)
Kornyushyna
19
DNA
O
HN
N
HO
N
O
HN
NH 2
HN
N
∗
N
O
dR O
∗
N
triketo
O
O
N
∗
N
∗
NH 2
N
dR O
imine enol-2
NH 2
N
HN
N
O
dR O
imine enol-1
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 indicates
the 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
Ia:G
Gh:G
Sp:G
20)
Oz:G
20.3
kcal/mol
16.3 kcal/mol
(Fig.9 Table 2)
3
Oz:G
Ia:G
2
Ia:G
DNA
Oz >
20, 26)
Gh/Ia > Sp
Oz:G
Sp
Gh/Ia
sp3
DNA
sp3
DNA
DNA
Sp
(melting temperature Tm
)
DNA
20 ºC
35)
Sp
DNA
DNA
2'C1'
C:G
36)
21.0 kcal/mol
DFT
25.0 kcal/mol
B3LYP/6-31G**
4.0 kcal/mol
C1'
3
21
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
4
Ia1:G-Ia8:G
Gh3:G
Ia1:G
21.4 kcal/mol
24.1 kcal/mol
Ia
Gh1:G-Gh16:G
Gh
Ia1:G
Gh/Ia
Gh
Ia
Sp:G
Sp2:G
Sp1:G
19.9 kcal/mol
Ia:G
Sp:G
Sp:G
Ia:G
Sp
Gh/Ia
26)
Oz:G
16.3 kcal/mol
Gh/Ia
Sp
Ia:G
Kino
Kornyushyna
Oz:G
Ia:G
Oz
20, 26)
Gh:G
Sp:G
DNA
Kino
DNA
23
Kornyushyna
3
DNA
(
Molecules, 19, 11030-11044 (2014)
)
1
G:C-C:G
Oz
Gh/Ia Sp
G:Oz G:Gh G:Ia G:Sp
G:Ia > G:Sp > G:Gh > G:Oz
1
2
DNA
Oz
Gh/Ia
Oz
Gh/Ia
Sp
Sp
20, 26)
20, 26)
DNA
DNA
DNA
DNA
DNA
DNA
37, 38)
DNA
DNA
DNA
DNA
DNA
DNA
(Fig.10)
DNA
DNA
DNA
39)
DNA
39)
DNA
37)
DNA
sp3
DNA
G:Ia
24
Fig.11
G:Sp
G:Oz
sp3
Fig.10 DNA synthesis by DNA polymerases. (A) DNA synthesis of undamaged DNA is
efficient. (B) DNA synthesis of damaged DNA is inefficient.
25
A
B
O
N
N
dR
H
O
N H
H
N
NH
O
H N
O
N
N
dR
N
N
N H
C
H
NH O
N
N H N
∗
dR
N
NH 2
N H O
dR
N
HN dR
H
H N
O
N
H
N H N
N
N H
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.
Sp
sp3
DNA
Oz
Ia
Ia
Sp
DNA
DNA
DNA
DNA
20, 26)
Oz > Gh/Ia > Sp
G:Ia
G:Gh
Ia
Ia
G:Oz
G:Ia G:Sp
20, 26)
DNA
DNA
hPol β-DNA
40)
3
DNA
26
2
2-1.
(PDB entry: 1BPY) 40)
hPol β
S-Ia R-Ia S-Sp R-Sp)
1
G:X
(X = Oz
hPol β-DNA
DNA
5'
T
-
Oz
20, 21)
dATP
dCTP
5
T
dATP
hPol β-DNA
dCTP
3'
G
G:C
2
G:X
DNA
G:X
9.0
T
Macromodel
OPLS2005/water
2-2. ab initio
Macromodel 9.0
G:X
2
C1'
C1'
(Fig.12) 20) 2-1
B3LYP/6-31G**
C1'
2
“A1T1”
1
N
“G2X2” “G3C3”
“A1T1”
(Fig.12 Fig.15)
Gaussian 03
30)
78.39)
G:X
Onsager reaction field
“A1T1”
“G3C3”
“A1T1 + G3C3”
(ΔE1
27
ΔE3 ΔE1+3)
(2)–(4)
(ε =
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. et
al., Molecules, 19, 11030-11044 (2014) with permission from MDPI.
28
2-3.
5
(A1 T1 G2 G3 C3)
N3 (xN3, yN3, zN3)
C5 (xC5, yC5, zC5) N1 (xN1, yN1, zN1)
C5N1
Pn
C5N3
2
(Fig.13A)
(5)
(Fig.13B) A1 T1 G2 G3 C3
Pn
n
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
G2
G3))
G2
A1 (θ (G2–A1))
C3 (θ (G2–C3))
G3
G2
T1 (θ (G2–T1))
C3 (θ (G3–C3))
(
A1
T1 (θ (A1–T1))
6
G2
G3 (θ (G2–
(Fig.14)
(6)-(11))
Fig.14 Calculated dihedral angle θ (G2–A1), θ (G2–T1) and θ (A1–T1) showed red arrows, and the
calculated 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
δ1
δ3
(12)
(13)
δ1 = θ (G2–A1) + θ (G2–T1) + θ (A1–T1)
(12)
δ3 = θ (G2–G3) + θ (G2–C3) + θ (G3–C3)
(13)
31
3
3-1. G:X
1
DNA
2
(Oz
Ia
sp3
Sp
“G:X (X = Oz
Ia Sp)
S
S-Ia R-Ia S-Sp R-Sp)”
>> G:R-Sp > G:S-Sp >> G:Oz
DNA
DNA
(PDB entry: 1BPY) 40)
hPol β
hPol β-DNA
G:X
G:S-Ia > G:R-Ia
2
G:X
1
R
G:X
G:X
2
2
hPol β-DNA
hPol β-DNA
G:X
hPol β-DNA
G:X
5'
(Fig.12 Fig.15) Fig.12
X
5'
A:T
A1T1 G2X2 G3C3
“A1T1”
X
3'
G:X
3'
“G2X2”
“G3C3”
Fig.15
3
DNA
X2
X2
X2
A1T1
DNA
32
G3C3
G2 X2
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. et
al., Molecules, 19, 11030-11044 (2014) with permission from MDPI.
33
3-2. X
5'
3'
DNA
DNA
“G2X2”
3-2-1. X
X2
5'
5'
“A1T1”
(ΔE1)
(2)
ΔE1DFT
(Table 3)
ΔE1SCRF
ΔE1DFT
ΔE1SCRF
R-Sp > S-Sp > S-Ia > Oz >
R-Ia
Kornyushyna
Gh/Ia
Sp
26)
Ia
5'
Sp
Oz
“A1T1”
Kornyushyna
R-Ia
ΔE1
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. et
al., 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'
“A1T1”
Ia
Oz
DNA
20)
3'
“G3C3”
3-2-2. X
3'
X2
3'
“G3C3”
ΔE3DFT
(
ΔE3SCRF)
(3)
(Table 3)
ΔE3DFT
X2
ΔE3SCRF
3'
“G3C3”
Oz
“G3C3”
X2
3'
“G3C3”
Oz
Ia
Sp
Oz
20, 26)
DNA
S-Sp
“G3C3”
R-Ia
“G3C3”
X2
3-2-3. G2X2
X2
5'
3'
X
5'
3'
G2 X2
“A1T1”
ΔE1+3DFT
(
Sp
Sp
ΔE1+3DFT
ΔE1+3DFT
S
“A1T1 + G3C3”
“A1T1 + G3C3”
(4)
ΔE1+3SCRF
Oz
ΔE1+3SCRF
Ia
ΔE1+3DFT
R
> S-Sp~R-Ia > S-Ia > Oz ΔE1+3SCRF
DNA
“G3C3”
ΔE1+3SCRF)
(Table 3)
Ia
3'
20, 26)
“G3C3”
Oz
R-Sp > R-Ia > S-Sp > S-Ia > Oz
R-Sp
R-Sp > S-Sp > R-Ia > S-Ia > Oz
“A1T1 + G3C3”
DNA
35
DNA
DNA
39)
DNA
DNA
DNA
DNA
37)
Ia
Sp
Oz
DNA
20, 26)
R-Ia
S-Sp
ΔE1+3DFT
26)
20, 26)
DNA
DNA
ΔE1+3SCRF
DNA
ΔE1+3SCRF
R-Ia
S-Sp
26)
in vivo
S-Sp
S-Sp
R-Sp
R-Sp
41)
X2
“G3C3”
5'
G:Oz
“A1T1”
G:Ia G:Sp
X2
1
3'
DNA
“A1T1 + G3C3”
G:X
DNA
Oz
S-Ia R-Ia S-Sp R-Sp
“A1T1”
DNA
3-3. G:C
Fig.15
DNA
Oz
S-Ia R-Ia S-Sp R-Sp
(12)
1
(13)
3-3-1. 5'
36
DNA
“G3C3”
G2 X2
5'
(θ (G2–T1)) A1
G2
T1 (θ (A1–T1))
(Fig.14
3
T1
(6)-(8)
)
(δ 1 )
(12)
θ (G2–A1)
d1
S-Sp > R-Sp > S-Ia > Oz >
θ (G2–T1) θ (A1–T1)
C > R-Ia
A1 (θ (G2–A1)) G2
(Table 4)
R-Ia
δ1
“A1T1”
(Table 4
Fig.15A
ΔE1
> R-Ia
15D)
R-Ia
ΔE1
δ1
S-Ia > Oz
“A1T1”
δ1
X2
R-Sp
S-Sp
ΔE1
Ia
δ1
ΔE1
R
ΔE1
S-Ia
(Fig.15C 15E) X2
Sp
S-Sp
S
C1'
5'
“A1T1”
S
R
20, 26)
δ1
ΔE1
Table 4 Dihedral angles θ (G2–A1), θ (G2–T1), and θ (A1–T1) (red arrows in Fig.14), and the
degree 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)
δ1
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
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
5'
(G2–C3))
G3
(3-3-1)
C3 (θ (G3–C3))
(Fig.14
G2
3
(δ 3 )
(13)
θ (G2–C3) θ (G3–C3)
θ (G2–G3)
δ3
R-Ia > R-Sp > S-Sp > S-Ia
(Table 5)
X2
3'
δ3
Oz
δ1
“G3C3”
(Table 5
ΔE3
S-Sp > S-Ia > Oz
G3C3
Fig.15A
15B)
3.3.1
δ3
δ1
G2 X2
δ1
R-Ia
15F)
C3 (θ
(9)-(11)
)
> C > Oz
G3 (θ (G2–G3)) G2
R-Sp
R-Ia
Ia
δ3
C1'
R-Sp
Sp
R
S
“G3C3”
C3
R-Ia
δ3
(Fig.15D
R-Sp
C1'
20, 26)
δ1
ΔE3
Table 5 Dihedral angles θ (G2–G3), θ (G2–C3), and θ (G3–C3) (blue arrows in Fig.14), and the
degree 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)
δ3
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°
a
X = the damage contained in the minimized structure.
39
3-3-3.
5'
X2
3'
5'
3'
δ1
(“δ1 + δ3”)
δ3
“ δ1 + δ3 ”
Table 6
> S-Sp > R-Ia > S-Ia > C > Oz
ΔE1+3
“ δ1 + δ3 ”
Oz
C
Oz
ΔE1+3
“G2C2”
R-Sp
“G2Oz2”
DNA
“A1T1 + G3C3”
“A1T1 + G3C3”
“ 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.
Xa
a
3-3-4.
3-3-3
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.
C
“G2Oz2” “G2C2”
“δ1 + δ3” “A1T1 + G3C3”
“ δ1 + δ3 ”
C
40
“ δ1 + δ3 ”
C
35.4°
C
“G2Oz2”
Oz
S-Ia
R-Ia
“ δ1 + δ3 ”
“A1T1 + G3C3”
“ δ1 + δ3 ”
4.0°
“G2S-Ia2”
“A1T1 + G3C3”
30.7°
“G2R-Ia2”
Fig.15
C
S-Sp
R-Sp
“G2R-Sp2”
“ δ1 + δ3 ”
81.1° 103.3°
“A1T1 + G3C3”
“G2S-Sp2”
“G2C2”
Fig.15
“G2C2”
“G2X2”
“ δ1 + δ3 ”
δ1
“A1T1 + G3C3”
C
δ3
S-Sp >
δ1
R-Ia > R-Sp > S-Ia > Oz δ3
R-Ia > R-Sp > S-Sp > Oz > S-Ia
C
δ3
δ1
“ δ1 + δ3 ”
δ3
δ1
δ1
δ1
“ δ1 + δ3 ”
δ3
DNA
δ3
20, 26)
“G2X2”
ΔE1+3
“A1T1 + G3C3”
DNA
G:Oz
DNA
DNA
41
4
DNA
Oz
Ia
Sp
20, 26)
G:Oz
2
G:Ia G:Sp
DNA
3
“G2X2”
DNA
“G2X2”
5'
X2
“A1T1”
R-Ia > Oz > S-Ia > S-Sp > R-Sp
“G3C3”
X2
3'
Oz > S-Ia > S-Sp > R-Ia > R-Sp
20, 26)
“G3C3”
“A1T1”
“A1T1 + G3C3”
“A1T1 + G3C3”
Oz > S-Ia > R-Ia > S-Sp > R-Sp
X2
3'
5'
DNA
20, 26)
DNA
X2
5'
δ1
3'
δ3
“A1T1” “G3C3”
20, 26)
3'
X2
“ δ1 + δ3 ”
“ δ1 + δ3 ”
G2C2
DNA
R-Sp >> S-Sp >> R-Ia > S-Ia >> Oz
“ δ1 + δ3 ”
“A1T1 + G3C3”
DNA
DNA
G:Oz
G:Ia G:Sp
DNA
G:Oz
DNA
G:Ia
42
G:Sp
5'
DNA
G:Oz
G:Ia
G:Sp
DNA
DNA
DNA
DNA
DNA
DNA
43
DNA
4
Oz
(
Chem. Res. Toxicol., 28, 1307-1316 (2015)
)
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 by
eukaryotic B- and Y-family DNA polymerases. Chem. Res. Toxicol., 28, 1307-1316. (DOI:
10.1021/acs.chemrestox.5b00114)”. Copyright 2015 American Chemical Society.
1
1
20, 26)
DNA
G:C-C:G
Gh/Ia
Oz
3
Oz
DNA
Sp
DNA
Oz
G:C-C:G
G:C-C:G
Oz
DNA
DNA
DNA
5
15
DNA
DNA
Pol δ
Pol α
Pol ε
3
(Table 7)
42, 43)
Pol γ Pol δ Pol ε
DNA
DNA
42-44)
DNA
Pol α
42, 43)
42)
46)
DNA
DNA
Pol δ
Pol ε
B
Pol α45)
Pol δ
Pol γ
Pol α
Pol ε
A
DNA
(Table 7) 43)
DNA
DNA
DNA
44
DNA
DNA
DNA
Pol ζ
DNA
Pol ζ
42, 43, 48)
B-
Pol η
Pol η
(Table 7)
Pol ι
Pol κ REV1
43, 47, 48)
Y
DNA
DNA
DNA
DNA
DNA
1
Pol α
Pol γ Pol ε Pol η
20, 21)
Pol γ
Oz
Pol α
Pol ε
Oz
Pol η
DNA
Oz
Oz
Oz
Pol κ REV1
DNA
(Table 7) 42, 43, 48, 49) Oz
DNA
Pol ι
DNA
Pol δ Pol ζ Pol ι Pol κ REV1
DNA
Oz
Table 7 The function of DNA polymerases 42, 43).
function
DNA polymerase
family
Pol γ
A-family
Pol α
Replicative 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
2
2-1.
20)
DNA
Oz
(5'-CTCATCAACATCTTXAATTCACAATCAATA-3'
(Fig.16)
Gh
30-mer
30-mer
X = Oz)
(X = Gh)
22)
DNA
(Fig.17)
8-oxoG
30-mer
(X = G
8-oxoG
THF)
(5'-*TATTGATTGTGAATT-3')
Alexa680
(Saitama, Japan)
Fig.16 Outline of the preparation of 30-mer DNA containing the Oz lesion.
O
O
H
O
Fig.17 Structure of THF.
46
THF
5'
2-2. DNA
yeast Pol ζ (yPol ζ)
yeast REV1 (yREV1)
Human Pol (hPol) δ
4
Enzymax (Lexington, KY)
(p125
p50
POLD1 POLD2 POLD3 POLD4
p66
p12)
p125
(Fig.18) C
6×His
p125
pTriEx-1.1/p125-His6
pTriEx-1.1/p125-His6
(Fig.19A)
Rosetta2 (DE3) pLysS
25 ºC
ºC
1 mM IPTG
3
-80
lysis
(20 mM sodium phosphate (pH 7.4),
0.5 M NaCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride (PMSF))
HisTrap HP
1 ml (GE Healthcare, Tokyo, Japan)
P40
(20 mM sodium phosphate
(pH 7.4), 0.5 M NaCl, 40 mM imidazole, 0.5 mM PMSF)
P500
(20
mM sodium phosphate (pH 7.4), 0.5 M NaCl, 500 mM imidazole, 0.5 mM PMSF)
p125
vivaspin 15 (Sartorius, Tokyo, Japan)
hPol δ
(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)
-80 ºC
50)
hPol δ
Fig.19)
p125
pTriEx-1.1/p125 N
FLAG
p12
p50
pCOLADuet-1/p50-p66-p12
pCOLADuet-1/p50-p66-p12
(DE3) pLysS
(Fig.18
16×His
p66
(Fig.19B)
pTriEx-1.1/p125
2
Rosetta2
1 mM IPTG
16 ºC
-80 ºC
p125
-80 ºC
47
12
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
GST
83 kDa
hPol ι
(Fig.20)
N
pGEX6P1/hPol ι
pGEX6P1/hPol ι
Rosetta2 (DE3) pLysS
25 ºC
-80 ºC
1 mM IPTG
3
binding
(PBS (pH 7.3); 140
mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.5 mM PMSF)
GSTrap HP 1 ml
(GE Healthcare)
binding
PG
(pH 7.4), 10 mM reduced glutathione, 0.5 mM PMSF)
vivaspin 15 (Sartorius)
(50 mM Tris-HCl
hPol ι
hPol ι
(50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol (2-ME), 0.5 mM
PMSF)
-80 ºC
Fig.20 Expression and purification of hPol ι.
49
hPol κ
6×His
POLK
99 kDa
hPol κ
N
pET15b/hPol κ
(Fig.21) pET15b/hPol κ
Rosetta2 (DE3) pLysS
25 ºC
-80 ºC
1 mM IPTG
3
lysis
HisTrap HP 1 ml (GE
Healthcare)
P40-T
(20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 40
mM imidazole, 0.01% Triton X-100, 0.5 mM PMSF)
P100-T
(20 mM sodium phosphate (pH 7.4), 0.5 M NaCl, 100 mM imidazole, 0.01% Triton X-100, 0.5
mM PMSF)
P200-T
(20 mM sodium phosphate (pH 7.4), 0.5
M NaCl, 200 mM imidazole, 0.01% Triton X-100, 0.5 mM PMSF)
vivaspin 15 (Sartorius)
hPol κ
hPol κ
(50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50% glycerol, 1 mM DTT, 0.5 mM PMSF)
-20 ºC
Fig.21 Expression and purification of hPol κ.
50
Human REV1 (hREV1)
REV1
138 kDa
REV1
REV1
REV1
51)
hREV1(341-829)
65 kDa
N
(Fig.22)
6×His
25 ºC
Pol δ
829
hREV1(341-829)
hREV1(341-829)
pET15b/ hREV1(341-829)
Rosetta2 (DE3) pLysS
341
(Fig.23)
pET15b/ hREV1(341-829)
1 mM IPTG
3
-80 ºC
hREV1(341-829)
(50 mM HEPES-NaOH
(pH 7.5), 0.5 M NaCl, 10% glycerol, 10 mM 2-ME, 0.5 mM PMSF)
-80 ºC
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
(62.5 mM Tris-HCl (pH 6.8), 10% glycerol,
2% SDS, 0.05 mg/ml bromophenol blue)
5
95 ºC
CBB Stain One (nacalai tesque, Kyoto, Japan)
SDS-PAGE
LAS3000 (Fujifilm,
Tokyo, Japan)
2-4.
CBB
(62.5 mM Tris-HCl
(pH 6.8), 10% glycerol, 2% SDS, 0.05 mg/ml bromophenol blue)
SDS-PAGE
10% methanol)
5
95 ºC
(25 mM Tris, 189 mM glycine,
Immobilon-P transfer membrane (Merck Millipore, Darmstadt, Germany)
Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA)
1
1
5%
0.1% PBS-T (137 mM NaCl,
2.7 mM KCl, 8.1 mM Na2HPO4•12H2O, 1.47 mM KH2PO4, 0.1% Tween 20)
1
0.1% PBS-T
Immobilon Western
chemiluminescent HRP substrate (Merck Millipore)
(Fujifilm)
1
2
LAS3000
Anti-His-tag (MBL, Nagoya, Japan)
Anti-DNA
polymerase δ p125 catalytic subunit (MBL) Anti-DNA polymerase δ p50 small subunit (MBL)
DNA
δ
p66
(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)
2
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 (BECKMAN
COULTER, Brea, CA)
53
2-3. DNA
DNA
p125
hPol δ
50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, 2 mM DTT, 100 µg/mL bovine
serum albumin (BSA)
yPol ζ
hPol κ hREV1(341-829)
yREV1
50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 5 mM DTT, 100 µg/mL BSA
hPol ι
40 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 150 mM NaCl, 1 mM DTT, 10 µg/mL BSA, 10%
glycerol
hPol δ
100 fmol
yPol ζ hPol ι hPol κ hREV1(341-829) yREV1
DNA
50 fmol
p125
100 fmol
5'
Alexa680
DNA
50 fmol
[32P]
dNTP
DNA
5 µl
30 ºC
30
30
p125
hPol ι
5 µl
30 ºC
stop
8M
15
5'
DNA
dNTP
Fig.24
hPol δ
yPol ζ yREV1
hPol κ
37 ºC
(15 mM EDTA, 10% glycerol)
16%
1×TBE (89 mM
Tris, 89 mM boric acid, 2 mM EDTA)
2.5 µl
5'
30 W
90
Alexa680
Imaging System (LI-COR, Lincoln, NE)
Odyssey® Infrared
5'
[32P]
BAS2500 bioimaging analyzer (Fujifilm)
54
Fig.24 Experimental design of DNA polymerase assay. The 15-mer primer is labelled with
Alexa 680 or [32P] indicated by ‘*’.
2-4.
16)
hREV1(341-829)
G
Oz THF 8-oxoG Gh
DNA
dCTP
4-20%
dCTP
dCTP
DNA
2-3
2-3
30 ºC
1
Km
Vmax
3
55
2
DNA
2
3
3-1. hPol δ
Oz
DNA
Pol δ
Pol α
Pol ε
B
DNA
hPol δ
DNA
p125
p50
p66
Oz
20)
Oz
4
DNA
hPol δ
Pol α
Pol ε
dGTP
4
4
52)
p125
p125
p12
p125
hPol δ
CBB
(Fig.25
Fig.26)
hPol δ
Fig.25 Expression and purification of recombinant p125. (A) p125 was electrophoresed and
stained 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 by
immunoblotting with anti-p125, anti-p50, anti-p66, anti-FLAG antibody.
p125
Oz
30-mer
DNA
(Fig.27A
p125
(Fig.27B
(Fig.27B
1)
7)
Oz
dGTP
p125
DNA
dGTP
(Fig.28A
(Fig.28B
Pol ε
dCTP
hPol δ
30-mer
Pol α
G
20)
5)
7)
Oz
dGTP
57
Oz
5)
hPol δ
Oz
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 the
presence 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 in
lanes 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 contained
no enzyme and are negative controls. Copyright 2015 American Chemical Society.
59
p125
3'-5'
hPol
DNA
δ
DNA
53)
G
DNA
DNA
Oz
3'-5'
(Fig.28A
6-9
Fig.28B
5
1-4
Oz
Fig.28A
)
1
hPol δ
Fig.28B
3'-5'
DNA
Oz
Oz
dGTP
G:C-C:G
Oz
DNA
20, 21)
dGTP
Oz
2
G
hPol δ
dGTP
hPol δ
G:C-C:G
60
Oz
Oz
DNA
3-2. yPol ζ
Oz
DNA
Pol ζ
DNA
UV
47, 54, 55)
DNA
DNA
Pol ζ
(6-4)
56)
DNA
O6 -
8-oxoG
(O6-methylguanine O6-MeG)
DNA
57)
THF
(cyclobutane pyrimidine dimer CPD) (Fig.29)
56, 58)
DNA
yPol ζ
Oz
DNA
O
O
HN
O
NH
N
N
dR
dR
O
Fig.29 Structure of CPD.
yPol ζ
Oz
G
5-7
(Fig.30A
DNA
1-3
hPol δ
(Fig.30A
)
DNA
G
DNA
DNA
yPol ζ
DNA
DNA
20, 21)
DNA
Oz
yPol ζ
5-7)
Oz
Oz
Pol ζ
DNA
G
G
dCTP
61
(Fig.30B
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) and
each 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 and
nucleotide 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 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.
62
1)
dGTP
dTTP
(Fig.30B
18-mer
3)
(Fig.30C
(Fig.30B
DNA
Oz
2)
Oz
5'
T
Oz
G
DNA
20-mer
(Fig.30C
(Fig.30C
dGTP
dGTP
dATP
18-mer
3)
1
Oz
dCTP
dTTP
4)
30E)
Pol ζ
4) dATP
20-mer
Oz
(Fig.30D
2
Pol ζ
G
Oz
58)
THF
8-oxoG
O6-MeG
57)
DNA
Pol ζ
DNA
Pol ζ
DNA
Pol ζ
47, 56, 59-61)
DNA
yPol ζ
Oz
DNA
G
DNA
yPol ζ
DNA
Oz
Oz
yPol ζ
63
dCTP
DNA
3-3. hPol ι
Oz
DNA
Pol ι
DNA
DNA
1
in vitro
G
62-65)
dTTP
Pol ι
2
66)
3
hPol ι (Fig.31)
DNA
G
DNA
dCTP
(Fig.32A
(Fig.32A
3, 5)
dTTP
2
2)
hPol ι
G
3
hPol ι
DNA
Fig.31 Expression and purification of recombinant hPol ι. (A) hPol ι was electrophoresed and
stained 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 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.
Oz
Pol ι
dATP
G
(Fig.32B
4, 5)
dTTP
G
(Fig.32B
Oz
6)
dGTP
DNA
dCTP
(Fig.32B
3)
DNA
dTTP
hPol ι
DNA
hPol ι
Y-family
hPol η
dGTP
67)
hPol ι
21)
dATP
hPol ι
hPol η
hPol ι
dGTP
dATP
Oz
(Fig.32B
2)
hPol ι
8-oxoG
8-oxoG
THF
68)
DNA
THF
DNA
hPol η
DNA
Oz
DNA
yPol ζ
65
THF
hPol ι
Oz
hPol ι
Oz
hPol ι
3-4. hPol κ
Oz
Pol κ
DNA
2
1
Pol κ
DNA
1
69-71)
2
DNA
DNA
72)
Wolfle
2
Pol κ
69-71)
dCTP
dGTP
dATP
dTTP
70)
hPol κ (Fig.33)
G
2
DNA
hPol κ
DNA
1
1)
dATP
29-mer
G
(Fig.34A
(Fig.34A
Oz
1)
(Fig.34B
1
(Fig.34B
4) dCTP
2)
dGTP
3-5)
DNA
(Fig.34B
(Fig.34A
dCTP
dTTP
30-mer
2
29-mer
G
Fig.34A
DNA
1
3)
)
Oz
dGTP
dATP
dTTP
(Fig.34B
(Fig.34B
Pol ι
Pol κ
Oz
DNA
DNA
DNA
66
2
DNA
5)
Fig.33 Expression and purification of recombinant hPol κ. (A) hPol κ was electrophoresed and
stained 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)
REV1
yREV1
Oz
Y
DNA
DNA
49, 73)
G
A
C
REV1
T
8-oxoG
N2-
dCTP
73-76)
hREV1
R357
dCTP
dCTP
77)
G
dCTP
REV1
dCTP
341
829
hREV1(341-829)
(Fig.35) DNA
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 immunoblotting
with anti-His antibody.
68
hREV1(341-829)
G
THF
8-oxoG
dCTP
(Fig.36B
36D
36E
3)
Oz
DNA
hREV1(341-829)
(Fig.36C
3)
Oz
dCTP
Oz
G:C-C:G
1
(Fig.36F
Oz
Gh/Ia
3)
hREV1(341-829)
dCTP
hREV1
Gh/Ia
dCTP
Oz
dNTP
G
Gh/Ia
Oz THF
G:C-C:G
8-oxoG
Gh/Ia
REV1
dNTP
5'
hREV1(341-829)
dCTP
(Fig.37A
(Fig.37A
4
dCTP
dGTP
4
6)
(Fig.37B
dGTP
hREV1(341-829)
(Fig.37D
G
37C 37E
6)
3)
G
3)
G
dTTP
8-oxoG
Oz
dTTP
5'
THF Gh/Ia
(Fig.37B
5'
dCTP
37C 37E
3)
5'
DNA
hREV1(341-829)
yREV1
hREV1
yREV1
(Fig.38B
Oz
Oz
Oz
THF Gh/Ia
dCTP
dCTP
3)
THF
(Fig.38B
3
Oz
38C
dCTP
G:C-C:G
69
dCTP
3
)
REV1
Oz
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, A
or 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
Oz
dCTP
REV1
DNA
Oz
dCTP
G
Oz THF 8-oxoG Gh/Ia
dCTP
dCTP
(Vmax/ Km)
(fins)
THF
(Table 8)
G
dCTP
fins
DNA
fins
0.19
(Table 8) THF
dCTP
G
8-oxoG
fins
6
DNA
Oz > THF
> 8-oxoG
Gh/Ia
8-oxoG
0.03 0.01
51)
Oz
19
dCTP
8-oxoG
>> THF > 8-oxoG
THF
REV1
fins
Oz
fins
0.12
(Table 8)
fins
DNA
dCTP
Oz > Gh/Ia > THF > 8-oxoG
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.
template
Vmax (µM•s-1)
Km (µM)
Vmax/Km (s-1)
finsa
G
1.33 × 10-2
2.06 × 10-2
6.45 × 10-1
1
Oz
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 × 10-2
0.03
8-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
72
REV1
dCTP
Oz
dCTP
Gh/Ia
hREV1
yREV1
THF 8-oxoG
DNA
(hREV1
G
R357 yREV1
R324)
dCTP
77, 78)
REV1
(hREV1
L358
yREV1
DNA
hREV1
H774
DNA
L325)
G
DNA
N7
G
G775 yREV1
M658
O6
G686
REV1
(Fig.39A)
77, 78)
G
8-oxoG
DNA
DNA
H7
hREV1
G
8-oxoG
51, 79)
dCTP
dCTP
dCTP
Howell
DNA
8-oxoG
(Fig.39B) 79) Km
H774
Km
DNA
REV1
Table 8
Gh/Ia > Oz
Km
dCTP
hREV1
Oz > Gh/Ia > 8-oxoG
8-oxoG
Oz
Oz
hREV1
Oz
G
N6
Oz
O6
G
(Fig.39C)
8-oxoG
(Fig.39D)
Oz
2
2
8-oxoG
Gh
hREV1
Gh
H774
hREV1
dCTP
hREV1
dCTP
8-oxoG
Gh
Gh
G775
Gh
Table 8
1
8-oxoG >
dCTP
8-oxoG
dCTP
Ia
(Scheme 1)
25)
Ia
73
Ia
Oz
pH
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 and
G775 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 et
al.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 hydrogen
bond. Copyright 2015 American Chemical Society.
74
2
Ia
(Fig.39E)
O7
Ia
Oz
Ia
hREV1
sp3
Oz
O7
Ia
hREV1
hREV1
dCTP
Gh/Ia
8-oxoG
dCTP
dCTP
Oz
hREV1
Gh/Ia
Gh/Ia
dCTP
Ia
Gh/Ia 8-oxoG
Oz
hREV1
hREV1
Oz
Oz
REV1
Oz
dCTP
REV1
NTH1
G:C-C:G
80)
Oz
Oz
dCTP
Oz:C
dCTP
REV1
Oz
Oz
dCTP
DNA
REV1
C
REV1
DNA
DNA
Pol ζ
81-85)
Pol ι Pol κ
Oz
8-oxoG
Oz:C
REV1
Pol δ
Gh/Ia
Oz
NEIL1
Oz:A
dCTP
REV1
Oz
Oz:G
G:C-C:G
REV1
Oz
G775
dCTP
DNA
75
DNA
Pol η
4
Pol δ
Pol ι Pol κ
Oz
DNA
Oz
(Table 9) Pol ζ
Oz
DNA
Oz
RVE1
Oz
Oz
G
DNA
(Table 9)
dCTP
(Table 9)
G:C-C:G
DNA
REV1
Pol ζ
Oz
DNA
DNA
DNA
Oz
DNA
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
Oz
20, 21)
REV1
DNA
dGTP
B
DNA
3
Oz
Oz
Oz
dGTP
2
G
DNA
DNA
Oz
Oz
77
dGTP
G
A T C
5
DNA
Oz
(
J. Nucleic Acids, 2014, 178350 (2015)
)
1
2
Oz
Gh/Ia Sp
3
DNA
DNA
DNA
20, 26)
Oz > Gh/Ia > Sp
Oz
DNA
2
Gh/Ia
3
G:C-C:G
Oz
DNA
Oz
20, 21)
Pol γ Pol κ Pol IV
Sp
KF exo-
4
Oz
Pol η
Pol ζ
DNA
Pol ι
Oz
G:C-C:G
(Pur:Pur)
(Pyr:Pyr)
Pur:Pyr
Pyr:Pur
DNA
DNA
DNA
DNA
(G:G
C:C C:A
)
86)
G:G > C:C > C:A
Beard
DNA
Pur:Pyr
78
Pyr:Pur
Pur:Pur
87)
Pyr:Pyr
DNA
20, 21)
4
REV1
DNA
Oz
Oz
G
A
C T
Oz:G
Oz:A
20)
DNA
Oz
Oz:G
Oz:A Oz:C Oz:T
Oz:G
Oz:G
Oz:A
Oz:C
Oz:T
Oz
Oz
3
DNA
(
KF exo-
Pol β
Pol η)
Tm
79
DNA
2
2-1.
DNA
30-mer
DNA
(5'-CTCATCAACATCTTXAATTCACAATCAATA-3' X = Oz)
9-mer
DNA
20)
(5'-TGCTXGCGT-3' X = Oz)
DNA
(5'-CTCATCAACATCTTGAATTCACAATCAATA-3')
5'
(5'-TATTGATTGTGAATTN-3' N = C
G A T)
9-mer DNA
T
5'-ACGCNAGCA-3'
Alexa680
(5'-TGCTNGCGT-3' N = C
N = C
G
A
T)
2-2. DNA
KF exo-
Fermentas (Waltham, MA)
hPol η
POLH
6×His
hPol β
CHIMERx (Milwaukee, WI)
78 kDa
hPol η
N
pET15b/hPol η
(Fig.40) pET15b/hPol η
Rosetta2 (DE3) pLysS
37 ºC
-80 ºC
4
1 mM IPTG
3
hPol δ
hPol η
(20 mM sodium phosphate (pH 7.4), 0.1 M NaCl, 1 mM EDTA, 10% glycerol,
10 mM 2-ME, 0.5 mM PMSF)
-80 ºC
80
Fig.40 Expression and purification of hPol η.
2-3. CBB
hPol η
(62.5 mM Tris-HCl (pH 6.8), 10%
glycerol, 2% SDS, 0.05 mg/ml bromophenol blue)
5
95 ºC
4
2-4.
4
Anti-His-tag (MBL)
2
1
Peroxidase-conjugated AffiniPure Goat Anti-Mouse
IgG (H+L) (Jackson ImmunoResearch)
81
2-5.
KF exo-
hPol β hPol η
KF exo-
DNA
50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT, 100 µg/ml BSA hPol
50 mM Tris-HCl (pH 8.8), 10 mM MgCl2, 1 mM DTT, 400 µg/ml BSA
β
hPol η
50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 5 mM DTT, 100 µg/mL
KF exo-
BSA
50 fmol
dATP
5'
hPol β hPol η
100 fmol
Alexa680
DNA
100 µM dNTPs (dCTP dGTP
dTTP)
DNA
DNA
Fig.41
hPol β hPol η
KF exo-
5 µl
37 ºC
30
8 M
5 µl
stop
16%
1×TBE
2.5 µl
30 W
60
Odyssey® Infrared Imaging System
(LI-COR Biosciences)
(14)
(Bandfull)
(Bandtotal)
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!(%) =
2-6. G
G
Oz
Band!"##
×100
Band!"!#$
(14)
5'
Oz
dNTP
nM DNA
Oz
G
10 nM
20
10 nM
5'-TATTGATTGTGAATTAC-3'
5'-TATTGATTGTGAATTAG-3'
DNA
Fig.42
100 µM
dNTP
(dCTP
dGTP dATP dTTP)
Fig.42 Experimental design of DNA polymerase assay for incorporation opposite 5'-neighboring
bases of G or Oz. The 16-mer primer is labeled with Alexa 680 indicated by ‘*’.
83
2-7.
Tm
9-mer
(5'-ACGCNAGCA-3'
DNA (5'-TGCTXGCGT-3' X = C
N = G
A
50 mM sodium phosphate (pH 7.0) 1 M NaCl
1:1
9-mer
DNA
4 µM
9-mer
(100 µl)
90 µl
5 min
(Life Technologies)
Ultrospec 3100 pro (GE Healthcare)
80 ºC
9-mer
C T)
60 ºC
20 ºC
T Oz)
1 ºC/min
260 nm
Tm
84
3
3-1. hPol β
Oz
hPol β
30
G
G
Oz
DNA
Oz
3'
16
4
G
DNA
hPol β
3'
3
C
(Fig.43A
7 10
1
4
)
G:T
(Fig.43A
Pur:Pyr
2
10
4
7
Fig.43C)
87)
Pur:Pur
Oz
DNA
Oz
(Fig.43B
G
4
1
7
8
10
)
(Fig.43D)
20)
Oz
hPol β Oz
Oz:G
Oz:G
G:G
(Fig.43B
4
43A
G:A
4
DNA
DNA
Pur:Pur
85
7
)
hPol β
Oz:G
Fig.43 Extension from primer ends by hPol β. Each primer contained a different nucleotide at
the 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 exoKF exo-
(Fig.44)
KF exo-
hPol β
G:C
(Fig.44A
1
4
7
10
3'
G:G
KF exo-
)
hPol β
G:C > G:T > G:A >
(Fig.44A, C)
20)
Oz
3'
(Fig.44B
Oz:G
4)
Oz:T
Oz:G
2
10
(Fig.44B
Fig.44D)
Oz:G
Oz:C
4
1
Oz:G
87
7
Oz:A
20)
Oz:A
Oz:A
Fig.44 Extension from primer ends by KF exo-. Each primer contained a different nucleotide at
the 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, and
10 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
KF exo-
hPol β
hPol η
G
Oz
hPol η
CBB
(Fig.45)
hPol β KF exo-
3'
(Fig.46A
1
G:C
4
7 10
)
exo-
hPol β
(G:G
50%
Fig.46C)
(Fig.46A
hPol η
hPol η
KF
G:A G:T)
4
7 10
DNA
88-90)
hPol η
Oz
20)
hPol η
DNA
88-90)
Oz:C
Oz:A Oz:T
4
1
DNA
Oz:G
3'
2
7
10
(Fig.46B
Fig.46D)
hPol β
Oz:G
KF exo-
Fig.45 Expression and purification of recombinant hPol η. (A) hPol η was electrophoresed and
stained 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 at
the 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, and
10 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
DNA
Oz:G
Oz:A Oz:C Oz:T
DNA
Tm
DNA
Oz
3-1
3-2 3-3
DNA
Tm
(Fig.47A
C:G
47B) A:T
C:G
55.1 ºC
48.9 ºC
(Table 10) (Fig.47C
(Table 10)
47D)
A:T
Oz:G
DNA
Oz:A
(Tm
Tm
45.7 ºC
Oz:C Oz:T
DNA
< 40.0 ºC)
Oz:G
(Table 10) (Fig.47E
Tm
(Table 10) (Fig.47G-L)
Oz:A
Oz:C Oz:T
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
< 40.0
Oz:C
< 40.0
Oz:T
< 40.0
91
Tm
47F)
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 of
absorbance 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
Oz
5'
hPol β
Oz:C Oz:T
KF exo- hPol η
Oz:A
Oz:G
DNA
hPol β
KF exo- hPol η
Oz:G
DNA
G
Oz
5'
DNA
G:C
Oz:G
G
DNA
dATP
(Fig.48A
48B
dGTP
hPol β
(Fig.48A
KF exo-
G
KF exo-
hPol β
3'-TTCT-5'
T
dGTP
4)
G
48B
3)
dTTP
hPol η
3-5)
88-90)
KF exo-
hPol β
dATP
(Fig.48A
48B
3'-TTCT-5'
dTTP
dATP
(Fig.48C
hPol η
Oz
T
hPol β
KF exo-
DNA
G
DNA
T
dGTP
hPol β
KF exo-
dCTP
94
(Fig.48C
Oz
hPol η
G
DNA
G
Oz
8-10)
Oz
9) G
hPol η
7)
T
(Fig.48C
DNA
3'-TT-5
3'-TT-5'
dATP
hPol η
Oz
G
DNA
Oz
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 of
primers 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
4
20, 21)
4
Oz
DNA
dGTP
DNA
2
Oz
Pol β
DNA
KF exo-
Pol β
Oz:C
Oz:G
Pol η
Oz:A Oz:C Oz:T
Oz:G
1
Oz:C
DNA
Oz:G
Oz:A Oz:T
Oz:A Oz:T
KF exo- Pol η
Oz:C
Tm
Oz:G
Oz:A Oz:T
DNA
Oz:G
Oz:G
20)
Oz:A
G:C-C:G
Oz
Oz:G
Oz:G
96
6
Oz
DNA
(
J. Biochem., 159, 323-329 (2016)
)
1
1
Oz
DNA
91, 92)
93)
K-ras
DNA
DNA
8-oxoG
Pol α
Pol β
8-oxoG
94, 95)
DNA
DNA
(5'-GG-3')
Iz
13)
(5'-IzIz-3')
11)
Oz
Iz
Oz
Iz
(5'-OzOz-3')
8-oxoG
Oz
4
Iz
Oz
DNA
Pol α
DNA
Oz
Pol δ Pol ε
1
DNA
DNA
Oz
Oz
1
DNA
KF exo-
calf thymus Pol (ctPol) α hPol β yPol ζ hPol η hPol ι hPol κ
97
yREV1
Oz
DNA
DNA
Oz
Oz
DNA
DNA
DNA
DNA
Table 11
Table 11 The function of DNA polymerases used in this chapter 42, 43).
DNA polymerase
Pol α
Replicative 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
2
2-1.
DNA
Oz
30-mer
(5'-CTCATCAACATCTXXAATTCACAATCAATA-3' XX = OzOz)
13)
(Fig.49)
6-mer
(5'-CTGGAA-3')
Iz
UV (365 nm)
6-mer
65 ºC
2
(5'-CTIzIzAA-3')
75
Oz
6-mer
11)
(5'-CTOzOzAA-3')
Oz
13-mer
6-mer
(5'-TTCACAATCAATA-3')
(New England Biolabs, Ipswich, MA)
Oz
5'
6-mer
(5'-CTCATCAACAT-3')
T4 DNA
5'
3'
T4
11-mer
13-mer
(Takara Bio, Otsu, Japan)
30-mer DNA-RNA
(5'-TATTGATTgTGAATTGCAGATgTTGATGAG-3', “g”
)
HPLC
Yokohama, Japan)
2'-
Oz
30-mer
time-of-flight mass spectrometer (Bruker Daltonics K.K.,
Oz
30-mer
DNA
30-mer
CPD
(XX = GG
CPD)
(5'-*TATTGATTGTGAATT-3')
99
5'
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
KF exoyREV1
Fermentas
ctPol α
hPol β
CHIMERx
yPol ζ
Enzymax
hPol η
5
hPol ι
hPol κ
hPol κ yREV1
DNA
4
2-3. DNA
yPol ζ
hPol ι
hPol β hPol η
ctPol α
DNA
4
5
DNA
ctPol α
100
KF exo-
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
5'
ctPol α
100 fmol
DNA
50 fmol
Alexa680
dNTP
DNA
DNA
dNTP
Fig.50
37 ºC
8M
30
5 µl
5 µl
ctPol α
stop
16%
1×TBE
2.5 µl
30 W
90
Odyssey® Infrared Imaging System (LI-COR Biosciences)
Fig.50 Experimental design of DNA polymerase assay. The 15-mer primer is labelled with
Alexa 680 indicated by ‘*’.
101
3
3-1. Oz
30-mer
DNA
13)
Iz
6-mer
6-mer
Oz
(Fig.51A)
6-mer
(Fig.51B) Oz
6-mer
1.3%
5'
11-mer 3'
Oz
Oz
6-mer
13-mer
30-mer
(Fig.48C)
DNA
6-mer
Oz
30-mer
0.12%
Oz
6-mer
5'
30-mer
30-mer
DNA
DNA
30-mer
HPLC
RNA
HPLC
Oz
13-mer
DNA-RNA
Oz
DNA
11-mer 3'
30-mer
(Fig. 51C)
30-mer
DNA
102
DNA-RNA
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 exo-
ctPol α
KF exo-
ctPol α
hPol β
Oz
hPol β
DNA
Oz
DNA
DNA
KF exo-
ctPol α
1-3)
α
hPol β
Oz
(Fig.52A-C
KF exo-
DNA
hPol β
3'
Oz
1
(Fig.52A-C
KF exo-
ctPol α hPol β
KF exo8)
ctPol α
7)
3'
3'
Oz
Oz
Oz
1
8)
hPol β
(Fig.52E
dCTP
dGTP
(Fig.52F
KF exo-
hPol β
3'
Oz
dGTP
20)
dGTP
Oz
Pol α Pol β
(Fig.52D
dGTP
Oz
ctPol α
1
dATP
dATP
6
7)
KF exo-
5)
dGTP
(Fig.52E
dATP
3'
ctPol
Oz
DNA
DNA
43)
DNA
exo-
KF
Pol α Pol β
DNA
Oz
KF exo-
1
20)
Pol α
Pol β
Oz
DNA
DNA
DNA
Oz
DNA
104
DNA
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- (250
µU 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 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 the Oxford University Press.
105
3-3. hPol ι
hPol κ
hPol ι
yREV1
hPol κ yREV1
4
Oz
DNA
Oz
hPol ι
DNA
yREV1
1
Oz
hPol ι
yREV1
Oz
hPol ι
3'
53B
5'
G
3'
Oz
yREV1
dCTP
3) Oz
3'
(Fig.53A
Oz
53B
(Fig.53A
Oz
8)
hPol κ
DNA
(Fig.53C
1-3)
OzOz
DNA
(Fig.53C
hPol ι
Oz
3'
(Fig.53A
Oz
9-12)
dCTP
dGTP
yREV1
Oz
(Fig.53B
dATP dTTP
3'
9) Oz
5-7)
Oz
3'
Oz
(Fig.53A
53B
3-6
9-12
yREV1
)
4
hPol ι
dCTP
Oz
hPol ι
yREV1
Oz
Pol ι
Pol κ REV1
43, 96)
DNA
DNA
DNA
DNA
DNA
Pol ι
Pol κ
1
4
Pol κ REV1
Oz
Oz
Oz
DNA
Pol ι
5'
1
Pol κ REV1
DNA
Oz
DNA
Oz
3'
DNA
DNA
DNA
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 a
single 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 the
four 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
DNA
Pol η
43, 96)
DNA
88-90)
DNA
CPD
88)
DNA
1
21)
REV1
DNA
Oz
KF exo-
DNA
Pol α
Oz
Pol β
DNA
5-7)
CPD
(Fig.54A
hPol η
DNA
5-7
Oz
Oz
3'
dATP dTTP
dCTP
dGTP
5'
G
(Fig.54B
dATP
1-3
3'
(Fig.54A
hPol η
DNA
Oz
(Fig.54A
)
Pol κ
Pol η
hPol η
9-11
Pol ι
5'
5)
dCTP
dGTP
1-4) Oz
dTTP
(Fig.54B
6-9)
Oz
(Fig.54B
1-4
6-9
)
hPol η
Oz
DNA
CPD
DNA
Oz
hPol η
DNA
Pol η
Oz
3'
Oz
KF exo-
Oz
Pol η
Oz
5'
Pol α
Oz
Pol β
Pol ι
DNA
DNA
DNA
108
Pol κ
REV1
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 in
lanes 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 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 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
DNA
Pol ζ
43)
DNA
Oz
DNA
DNA
Oz
DNA
yPol ζ
Oz
DNA
(Fig.55A
5-7)
Oz
5-7
DNA
1-3
(Fig.55A
)
DNA
dCTP
17-mer
dTTP
G
3'
(Fig.55B
7) 3'
6
9
(Fig.55B
3'
Oz
Pol ζ
(Fig.55B
5'
Oz
1) dGTP
Oz
dCTP
2
dGTP
dTTP
(Fig.55B
9)
(Fig.55B
1
4
)
Oz
Pol ζ
Oz
6
dATP
(Fig.55B
Pol ζ
4)
8
3
)
1
DNA
DNA
UV
47, 54, 55)
4
DNA
G
Pol ζ
Oz
DNA
Oz
Oz
1
1
Pol ζ
Pol ζ
110
Oz
DNA
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 the
Oxford University Press.
111
4
KF exo-
Pol α
Pol β
Pol ι
REV1
Oz
3'
Oz
Oz
5'
(Table 12)
3'
Oz
Oz
Oz
DNA
Pol κ
Oz
Pol κ
3'
Oz
Oz
DNA
(Table 12)
Oz
DNA
DNA
DNA
DNA
CPD
DNA
DNA
Pol η
(Table 12)
Oz
Oz
3'
5'
Oz
Oz
DNA
Pol ζ
Oz
DNA
Oz
DNA
(Table 12)
Oz
DNA
DNA
Pol ζ
Oz
DNA
Oz
DNA
Pol ζ
DNA
Oz
DNA
Oz
dGTP
Oz
REV1
KF exo-
20, 21)
REV1
DNA
4
Oz
DNA
Oz
112
dGTP
Oz
Oz
G:C-C:G
Table 12 Summary of chapter 6.
Insertion
opposite 3' Oz
Extension
opposite 5' Oz
Replicative DNA polymerases
Pol α
G, A, C
stall
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 η
G, A, C, T
G, A, C, T
low efficiency
involved in translesion
Pol ι
G, A, T
synthesis
Pol κ
stall
stall
REV1
C
113
stall
7
K-ras
codon 12
G:C-C:G
G:C-T:A
K-ras
(Table 1)
G:C-C:G
Oz
Sp
DNA
Gh/Ia
Oz
8-oxoG
11-13)
6)
2
DNA
DNA
20, 26)
2
Oz
Gh/Ia
3
Oz
Sp
G:C-C:G
Gh/Ia Sp
DNA
2
Oz:G
3
Ia:G
sp3
Gh/Ia
Sp
Oz
DNA
Sp
sp3
DNA
Oz
Gh/Ia
Sp:G
DNA
DNA
DNA
DNA
DNA
Oz
DNA
G:C-C:G
Oz
4
6
Oz
Oz
Oz
DNA
REV1
REV1
DNA
KF exo-
DNA
114
5
Oz
DNA
Oz
G:C-C:G
Oz
REV1
DNA
DNA
Oz
Oz
DNA
DNA
Oz
DNA
DNA
DNA
DNA
NEIL1
Oz:C
Oz
Oz:C
Oz:A
80)
Oz:A
NTH1
Oz:G
Oz:G
Oz
Oz
Oz
Oz
Oz
REV1
Oz
Oz
DNA
DNA
DNA
(xeroderma pigmentosum
XP)
V
Pol η
115
97)
Pol η
CPD
DNA
DNA
Oz
REV1
REV1
ι
Pol ζ
Pol κ
Pol η Pol
DNA
81-85)
REV1
DNA
Oz
Oz
DNA
DNA
Oz
G:C-C:G
Oz
Oz
DNA
DNA
Pol ζ
mRNA
98)
REV1
mRNA
16
99)
DNA
Oz
Oz
in vitro
Oz
Oz
8-oxoG
Oz
Oz
DNA
Oz
1
116
18)
Oz
8-oxoG
Oz
Oz
Oz
Oz
Oz
Oz
Oz
DNA
Oz
Oz
Oz
117
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