DNA Repair and Mutagenesis of Reactive Oxygen Species ...
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DNA Repair and Mutagenesis of Reactive Oxygen Species-Generated Lesions Masaaki (Maki) Moriya SUNY at Stony Brook
Transcript of DNA Repair and Mutagenesis of Reactive Oxygen Species ...
DNA Repair and Mutagenesis of Reactive Oxygen Species-Generated Lesions
Masaaki (Maki) MoriyaSUNY at Stony Brook
ContributorsSUNYI.-Y. Yang, X. Liu, K. Hashimoto, R.L. Revine, S. Stein G. Pandya, G. ChanS. Attaluri, R. Rieger, M. C. Torres, S. Khullar, Y. Huang, F. JohnsonT. Zaliznyak, C. de los SantosH. Miller, A. Grollman
UMN Y. Lao, S. Hecht
OthersJ.E. Cleaver, M. Cordeiro-Stone, F. Hanaoka, J.H.J. Hoeijmakers, H. Ohmori, Z. Wang, R. Woodgate
Generation of DNA Adducts by Reactive Oxygen Species
Reactive oxygen species
DNA
Oxidized bases8-Oxo dG, dA2-OH dAThymine glycol5-OH-methyl dCetc
Lipid peroxidationEnals
Epoxides
Exocyclic propano and propeno adducts
α and γ-OH-propano dGsM1G, etc
Etheno adductsεdA, εdC, εdGSubstituted εdNetc
DNA DNA
dG in DNA
+
Bifunctional aldehyde(acrolein, crotonaldehyde)
+
F. Johnson, S. Khullar, Y. Huang(SUNY) S. Hecht, Y. Lao (UMN)
dRH
O
N
NN N
NOH
HO
HHN
N
N N
NO
OH
H dR
N
N
N N
NO
H dR
HO
γ-OH-PdG α-OH-PdG
R
OH
C
dR P
N
N N
NC
PdR
N
N
NN
N N
dR
HO
H
dR
R
R
CH3
Site-specific Procedure
1. Synthesis and purification of modified oligonucleotides
2. Incorporation into a vector
3. Introduction into a host cell
4. Recovery of progeny
5. Analysis for mutagenic and repair events
Obstacles to mutation studies
• DNA Repair• DNA synthesis block
Preferential replication of undamaged strand = Labor-intensive analysis for TLS events
Formation of DSBs→ NHEJ →Deletion mutants• Determination of targeted events
Site-specific mutagenesis approach
G
T
A
C
I.-Y. Yang, K. Hashimoto, X. Liu, R.L. Revine, S. Stein, G. Pandya (SUNY)
C. Lawrence
pMS2 pBT
C. Lawrence
Site-specific mutagenesis strategy
TACGTAATAXCT
TATNGAATAXCT
TACGTAATGCAT
TAC GTAATG CAT
SnaB I
-GGACTTTGTAGGATACCCTCGCTTT--GGACTTTGTGGGATACCCTCGCTTT-
AACACCCTATGGGA-32P
G A G A G A G A G A
Hybridization temp. = 4(G+C) + 2(A+T) - 4
K. Itakura, 1979
Site-specific mutagenesis approach
G
T
A
C
G
Hybridization
C A
T
I.-Y. Yang, K. Hashimoto, X. Liu, R.L. Revine, S. Stein, G. Pandya (SUNY)
C. Lawrence
pMS2 pBT
C. Lawrence
Oligonucleotide Probe hybridization
∆
A
C
G
T
dG in DNA
+
Bifunctional aldehyde(acrolein, crotonaldehyde)
+
F. Johnson, S. Khullar, Y. Huang (SUNY), S. Hecht, Y. Lao (UMN)
dRH
O
N
NN N
NOH
HO
HHN
N
N N
NO
OH
H dR
N
N
N N
NO
H dR
HO
γ-OH-PdG α-OH-PdG
R
OH
C
dR P
N
N N
NC
PdR
N
N
NN
N N
dR
HO
H
dR
R
R
Efficiency of translesion synthesis across monoadducts in human XPA cells
0
10
20
30
40
50
60
dG -OH-PdG -OH-PdG R Sγα
% o
f pro
g eny
f ro m
m
odi f i
ed s
t ran
d
α
Methyl- γ- OH-PdG
I.-Y. Yang, S. Stein (SUNY)
Mutagenicity of PdG adducts
T > C, A-----
11< 0.4
α-OH-PdGγ-OH-PdG
S isomerR isomer
Adduct
T > C, AT > A, C
105
HumanXPA cell
Miscoding Specificity
Miscoding Frequency (%)
Host
I.-Y. Yang, S. Stein (SUNY)
Genotoxic (point mutations) potency
= TLS efficiency x Fidelity
CH3- γ -OH (S) > CH3- γ -OH (R), α-OH >> γ – OH-PdG
HO
dRH
O
N
N
N N
N
N
N
N N
NOOH
H dR
γ(8)-OH-PdGα(6)-OH-PdG
S. Khullar, Y. Huang, F. Johnson (SUNY)
NN
dRO
NHH
H +N
N
NN
N O
dR
H
HO
α -OH-PdG(syn) dC+(anti)
HO
H
dR
O
N
N
NN
N
+H
HH N
dRNN N
N
dA+(anti)α -OH-PdG(syn)
H
O
H
O
OHH
dRN
NN
N
N HH N
O dR
NN
H
dC(anti)γ -OH-PdG(anti)
Non-mutagenic and mutagenic pairing
T. Zaliznyak, C. de los Santos (SUNY)
Extension from C terminus opposite adducts
5’ P-ATC3’ -TAXCTCCTCGTC
dG PdG α-OH-PdGγ-OH-PdGX =
←Xpol
Exo- Klenow
I.-Y. Yang, H. Miller (SUNY)
Incorporation of dNTP opposite a DNA adduct
dG PdG γ-OH-PdG α-OH-PdG
dNTP = A C G T A C G T A C G T A C G T
AXC
dNTP32P
X
Exo- Klenow
I.-Y. Yang, H. Miller (SUNY)
Sister chromatid cohesionXPol σ
DSB repair, BER?XPol λ
NHEJXPol µ
DNA cross-link repair APol θ
Translesion synthesisYREV 1Translesion synthesisBPol ζ
Translesion synthesisYPol ι
Translesion synthesisYPol κ
Translesion synthesisYPol η
DNA replication/repairBPol ε
DNA replication/repairBPol δ
mtDNA replication/repairAPol γ
Base excision repairXPol β
Priming DNA synthesisBPol α
Function(s)Pol familyName
Eukaryotic DNA polymerases
1.1022371XPV (CTag)
6.8*2417316XPV- mXPV
< 0.5000232XPV (XP30RO)
10.47417242XPA
MF(%)CATG
Host
Pol η contributes to α-OH-PdG→T mutations
* P < 0.001
α-OH-PdG →G, T, A, C
I.- Y. Yang (SUNY)
A C G T
A C G TN = A C G T
Incorporation
Extension
pol η
Y = A C G T
A C G T
pol ι REV1
A C G TA C G T
A C G T
pol ζ
A C G T
A C G T
XC
32 PY
4 dNTPs
XC
dNTP32 P
pol κ
In vitro translesion synthesis catalyzed by specialized polymerases
I.-Y. Yang, H. Miller (SUNY)
dG in DNA
+
Bifunctional aldehyde(acrolein, crotonaldehyde)
+F. Johnson, S.Khullar, Y. Huang (SUNY) S. Hecht, Y. Lao (UMN)
Model ICL
dRH
O
N
NN N
NOH
HO
HHN
N
N N
NO
OH
H dR
N
N
N N
NO
H dR
HO
γ-OH-PdG α-OH-PdG
R
OH
C
dR P
N
N N
NC
PdR
N
N
NN
N N
dR
HO
H
dR
R
R
X. Liu, I.-Y. Yang (SUNY)
C G1
G2 C
XPA, NER+: 2~3% T
NER+: ~0.7% T
Repair events in human cells
X. Liu (SUNY)
5’5’ C(G1)
(G2)C
C(G1)(G2)C
(G2)C(G1) G C/A
(G2)C(G1)
C(G1)(G2)C
Replication block
Incision
C(G1)(G2)C
TLS
TLS
NER
Replication-dependent and -independent ICL repair
Limitation of human cells as a host for mechanistic studies• Lack of various mutant cell lines
↓
Use of RNAi or gene KO mouse cells as a host
X. Liu (SUNY)
CGGC
Mutants
ERCC1 +/+
ERCC1 -/-
CGGC
Mutants
ColE1ampbsd f1SV40Polyoma TPoly
Location and size of deletionX. Liu (SUNY)
Plasmid map
Original size
5’5’ C(G1)
(G2)C
C(G1)(G2)C
C(G1)(G2)C
C(G1)(G2)C
C(G1)
Mus81/Eme1
(G2)C
C(G1)(G2)C
XPF/Ercc1
Deletions
X
Site-specific approach
Characterization of genotoxicity of DNA adducts
Clear relationship between cause and effect
Quite labor-intensive
Effects of sequence context on B[a]P-dG-induced G → T mutations
0
10
20
30
40
50
G1 G2
E. coliMonkey
5’ CTG1G2CC 3’
Freq
uenc
y of
G to
T, %
Moriya (SUNY)
