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)