Carlos de los Santos: TODAY’S SPEAKER - National … · 2012-05-02 · Carlos de los Santos:...

Carlos de los Santos: TODAY’S SPEAKER

Dan Bogenhagen: DNA replica6on and repair in mitochondria

Bruce Demple*: Oxida6ve DNA damage; BER; oxida6ve stress responses

Miguel Garcia‐Diaz*: DNA replica6on fidelity; protein structure; gene regula6on in mitochondria

Arthur Grollman: Mutagenesis and molecular carcinogenesis (aristolochic acid); structural biology

Francis Johnson & Charles Iden: Synthesis, purifica6on and analysis of single‐lesion DNA molecules

Orlando Schärer: Mechanisms of mammalian NER; DNA interstrand crosslinks

Kate Dickman: DNA damage and renal disease

Mark Lukin: genera6on of site‐specific DNA lesions Masaaki Moriya: translesion DNA polymerases

Mamta Naidu: Radia6on biology & BER; heavy ion studies

Thomas Rosenquist: gene6cs of aristolochic acid toxicology

*Recruited through Consor6um for InterDisciplinary Environmental Research

New insights into NER recognition: When bulky & perturbing is not enough

4/17/12

Kwong-Ngai Hung Serge Smirnov David Cullinam Zhen Lin Jenifer Bohon Raphael Hazel Omar Armstrong Tanya Zaliznyak Smita Mohanty Mahmoud El-Katheeb Kegui Tian Freda Sansaricq Dmitry Zamyatkin Kimberly Conlon Monica McTigue

Arthur Grollman Francis Johnson Charles Iden Carlos Simmerling Orlando Scharer Miguel Garcia-Diaz Masaaki Moriya Mark Lukin

NCI, NIEHS

DNA Damage Rate < Repair Rate

Healthy Genome

DNA Damage Rate > Repair Rate

Sick Cell

Cancer Ageing Repair

Diseases

Bulky Adducts Alkylating Agents

Ionizing, UV

Radiation

Chemical Degradation

Endogenous (ROS, LPO)

Exog

enou

s En

doge

nous

DSB

SSB

AP

SBD

ICl

Cl

DNA Damage and Repair

dRN

NNH

N

O

O

OH

O

dR

OOH

OH

OH

AP Sites

OH

OHOH

dA(N6) or dG(N2)

Benzo[a]Pyrene

NO2

2-Nitrofluorene

PtNH3

NH3

dG(N7)

dG(N7)

cis-platinum

N

NHNH

N

O O

OO

dR dR

Cyclobutane TT dimer 6-4 Photoproducts

NER Lesions

N

N

N

N N

O

OH

dG(N2)H

N

N N

N

NH2

O

OH

OHH

5’,8-cyclo-dA

Acrolein

NER Recognition of Bulky Adducts

Structure of a RAD4/RAD23-CPD-DNA Complex

Min, J-H & Pavletich, N.P (2007) Nature 449, 570-576

GG-NER Resistant DNA Lesions

dG-N2 Adducts (AAF, 3ABA)

dA-N6 Adducts (ALII)

NO2 2-Nitrofluorene (2-NF)

NH2 2-Aminofluorene (2-AF)

NHAc 2-Acetylaminofluorene (2-AAF)

Aminofluorene

NO2

2-NF NHAc

2-AAF

NAcOH

N-hydroxy-2-AAF

CYP1A2 CYP1A2 XO

NAcOSO3H

N-SO4-2-AAF

NHOSO3H

N-SO4-2-AF

NH2

2-AF

NHOH

N-hydroxy-2-AF

NHOAc

N-Acetoxy-2-AF

SO4 transferase SO4

transferase O-Acetylase

DNA-Adducts

N, O-Acetyl-transferase

NH N

NHN

N

O

NH2

dR

dG-C8-AF

NAc N

NHN

N

O

NH2

dR

dG-C8-AAF

NHAcN

NHN

N

O

N

dR

H

dG-N2-AAF

DNA-Adducts

dG-N2-AAF

• Is mutagenic: DNA-Pol I inserts dAMP, suggesting G → T mutations in bacteria

• It blocks mammalian Pol α activity in vitro, but TLS polymerases η and κ can bypass the lesion

• It induces G → T transversions in mammalian cells

• NER processing of dG-N2-AAF needs full study

• Is a minor guanine adduct

Mutation Research 442 1999. 9–18

• dG-N2-AAF is a persistent DNA lesion

NMR Constraints

Damaged DNA Sample

NMR Studies

Restrained MD

Structure Analysis and Validation

5’-d(C1 G2 T3 A4 C5 X6 C7 A8 T9 G10 C11)

d(G22 C21 A20 T19 G18 C17 G16 T15 A14 C13 G12)-5’

A & B, AAFH4 to G6H1’ and T19H1’ C & D, AAFH5 to G6H1’ and T19H1’

D2O NOESY

5’

3’ H1’

H1’

H6

H8

Zaliznyak, et al. (2006) Chem. Res. Toxicol. 19, 745-752.

5’-d(C1 G2 T3 A4 C5 X6 C7 A8 T9 G10 C11)

d(G22 C21 A20 T19 G18 C17 G16 T15 A14 C13 G12)-5’

H2O NOESY

C1’ C1’

T A

C G C1’

C1’


5’

5’

5’

5’

G6 G6

C17 C17

Refined MD Structure


Space Filling Models Major Groove

5’

3’

90°


Minor Groove

5’

3’

90°

Space Filling Models


Minor groove width (Å) 2.0-4.7 (5.9)

Minor groove depth (Å) 5.6-7.2 (4.6)

BP propeller twist (°) 1-34 (<3.7)

Base pair buckle (°) 2-18 (0)

AAF Exposed Surface 17%

T15C5’ A8C3’

A4C5’ T19C3’

Structural Parameters


Duplex Stability

45°C 15°C

20°C 50°C

25°C 60°C

14.0 13.0 12.0 11.0 10.0 PPM

30°C

14.0 13.0 12.0 11.0 10.0 PPM

65°C

10°C

T3,T9

T15

T19,G6

G2

G10,G16

G18 G6(N2H)

35°C

G6 G16

G18 G6(N2H)


30 50 70

Temperature (°C)

OD

260

0

0.5

1

1.5

2

1.7

1.9

2.1

2.3

(1/T

m) x

10-

2 (1

/°K)

-14 -13.5 -13 -12.5

ln(Ct/4)

Tm (°C) ∆H0

(kCal/Mol) ∆S0

(Cal/Mol°K) ∆G0

25°C (kCal/Mol)

∆G037°C

(kCal/Mol) AAF-Duplex 60.5 -93 ± 9 -262 ± 20 -14.9 ± 0.3 -11.8 ± 0.3 Control Duplex 54.3 -98 ± 11 -285 ± 23 -13.1 ± 0.3 -9.6 ± 0.3

dG-N2-AAF Duplex Stability


Adopts a regular helical structure with WC alignments

The lesion increases the duplex thermal and thermodynamics stability

The AAF moiety resides in the minor groove without protruding out of the helix boundaries

The minor groove deepens and narrows, sheltering AAF from water exposure

dG-N2-AAF Duplex


1 2 3 4 5

NER Repair of dG-N2-AAF

Lane 1: Hela whole cell extracts

Lane 2: Hela whole cell extracts + plasmid DNA containing a (1,3)-N7-N7 cis-Pt adduct

Lane 3-5: Three different preparations of Hela whole cell extracts + plasmid DNA containing a dG-N2-AAF lesion


Some bulky dG-(N2) adducts of defined topology cause minimal structural perturbations and increase duplex

stability.

Thus, they evade GG-NER processing, persist in cellular DNA and are likely to

have a prevalent role in chemical mutagenesis and disease.


Cosman, et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1914-1918. de los Santos, et al. (1992) Biochemistry. 31, 5245-5252. Zaliznyak, et al. (2006) Chem. Res. Toxicol. 19, 745-752.

(+) and (-) trans-benzo[a]pyrene-N2-dG Lesions AAF-N2-dG Lesions

dG-N2-AAF dG-N2-3ABA

Topology of helix-stabilizing lesions

3-Nitrobenzanthrone

O

NO2

3NBA

Present in coal fly ash, diesel engine exhausts and air particulate matter

Strong mammalian mutagen

Causes DSB and induces micronuclei formation in human cells

Causes squamous cell lung tumors in rats.

Epidemiologically linked to lung cancer in humans

N

NH

N

N

O

NH

O

NH2dR

O

NO2

O

NH2

N

N

N

N

dR

NH

O

NH2

N

O

N

NH

N

N

O

dRNH2

H

O

NHOH

O

NH+

O

NHOR

NAT1NAT2

SULT1A1SULT1A2

NQO1 NQO1CYPOR CYPOR

CYP1A1CYP1A2

SOLVOLYSIS

DNA DNA

DNA

3NBA 3ABA

dG(N2)-3ABA

dG(C8)-ABA dA(N6)-3ABA1

3 4

6

7

810

N

N

N

N

O

O

OH

OHNH

O

NO2

NO2

N

N

N

N

O

O

OH

DMTONH

O

NO2

NO2

N

N

N

N

O

O

O

DMTONH

O

NO2

NO2

PO

CN

N CH3

CH3

CH3

CH3

N

N

N

N

O

O

TBDMSO

TBDMSONH

O

NO2

NO2

N

NH

N

N

O

O

OH

OHNH

O

NO2

a.

b.

c. d.

Complexmixture ofproducts

c.

1 2

3 4 5

Scheme 1. Synthesis of protected dG(N2)-3ABA phosphoramidite

a. 1M tetrabutylammonium fluoride, THF; b. Triethylamine trihydrofluoride:triethylamine:DMF (4:3:6);c. Dimethoxytrityl chloride, pyridine; d. Cyanoethoxy diisopropylamino chlorophosphine.

Lukin, et al. (2011) J. Nucleic Acids Epub 2011 Nov 17.

N

NH

N

N

O

O

O

ONH

O

NH2

PO

O

P

OH

OO

OH

7

DNA

DNA

N

N

N

N

O

O

O

ONH

O

NO2

NO2

PO

O

P

OH

OO

OH

6

DNA

DNA

a. , b.

Scheme 2. Deprotection and nitro reduction of dG(N2)-NBA in oligonucleotides

a.1M tetrabutylammonium fluoride, THF; b. 1M Na2S.

G1 T2 A3 T4 X5 C6 C7 G8 G9 C10 A11 T12 A13 C14

Lukin, et al. (2011) J. Nucleic Acids Epub 2011 Nov 17.

dG-(N2)-3ABA Duplex

2.88

2.90

2.92

2.94

2.96

2.98

3.00

3.02

-13.5 -13.0 -12.5 -12.0 -11.5 -11.0

ln (C)

1000

/Tm

(°C

-1)

2.88

2.90

2.92

2.94

2.96

2.98

3.00

3.02

-13.5 -13.0 -12.5 -12.0 -11.5 -11.0

ln (C)

1000

/Tm

(°C

-1)

2.88

2.90

2.92

2.94

2.96

2.98

3.00

3.02

-13.5 -13.0 -12.5 -12.0 -11.5 -11.0

2.88

2.90

2.92

2.94

2.96

2.98

3.00

3.02

-13.5 -13.0 -12.5 -12.0 -11.5 -11.0

ln (C)

1000

/Tm

(°C

-1)

0.8

1.0

1.2

1.4

1.6

1.8

35 45 55 65 75 85

Temp (°C)AU

260

0.8

1.0

1.2

1.4

1.6

1.8

35 45 55 65 75 85

Temp (°C)AU

260

0.8

1.0

1.2

1.4

1.6

1.8

35 45 55 65 75 85

0.8

1.0

1.2

1.4

1.6

1.8

35 45 55 65 75 85

Temp (°C)AU

260

dG-(N2)-3ABA Duplex

Control Duplex

Control duplex

dG(2N)-ABA duplex -1.5

ΔΔG˚37/lesion, kcal/mol

-14.9 ± 0.3

-17.9 ± 0.8

ΔG˚37, kcal/mol

-18.2 ± 0.5

-21 ± 1.1

ΔG˚25, kcal/mol

-267 ± 11

-252 ± 24

ΔS˚25, cal/mol·K

-98 ± 4

-95 ± 8

ΔH˚25, kcal/mol

dG-(N2)-3ABA Duplex

dG-N2-AAF dG-N2-3ABA


Aristolochic Acids

Aristolochia elegans

O

O

O

OH

NO2

X

AAII : X = HAAI : X = OMeAAI : X = OMe

AAII : X = H

Aristolochia clematitis

AA-I is nephrotoxic: causes interstitial fibrosis with progression to end-stage renal disease

Many patients develop urothelial cancer

Both AA-I & AA-II are genotoxic, mostly leading to AT → TA transversions

The National Toxicology Program listed the AAs as established human carcinogens

AA-dA adducts persist in chromosomal DNA for several years

Aristolochic Acids

NH

N N

N

dRib

O

NH

O

O

O

NH

X

NHN

N

N

dRib

O

O

O

NH

X

N

O

O

O

OH

NO2

X

O

O

O

NHOH

X

XO, NQO1, CYP1A2, CYP1A1,

O-acetylation SO4

transferase

O

O

O

N

X

+

AL-dG

AL-dA

dA-(N6)-ALII Duplex

C1 G2 T3 A4 C5 X6 C7 A8 T9 G10 C11 G22 C21 A20 T19 G18 T17 G16 T15 A14 C13 G12

O

N

N N

N

N H

O

O

O

N H

T B S O

T B S O

O

O

O

N H

Br

BH O

N

N N

N

N H

O

O

O

N H

D M T O

O P

O C E

N

Attaluri et al. (2010) Nucleic Acids Res., 38, 339-352.

N

N N

N

N H

N H

O

O

O 2

4

8

9 10

11

dR

dA-(N6)-ALII Duplex

A) AAH11-AAH10 B) AAH10-AAH9 C) AAH8-AAH9

Lukin et al (2012) Nucleic Acids Res. 2012 40, 2759-2770.

D2O NOESY

dA-(N6)-ALII Duplex

6.5

6.0

5.5

ppm

C1

G2

T3

A4

C5

A6

C7

A8

T9

G10C11

C7*

C1*

C11*

8.5 8.0 7.5 7.0 ppm

6.5

6.0

5.5

ppm

G12

C13

A14

T15

G16

T17

G18

T19

A20

G22

C13* C21

C21*

5’

3’ H1’

H1’

H6

H8


C1 G2 T3 A4 C5 X6 C7 A8 T9 G10 C11

G22 C21 A20 T19 G18 T17 G16 T15 A14 C13 G12

H2O NOESY

G2 A20 A4 G18 G16 A8 A14

G10

Ter

dA-(N6)-ALII Duplex

C1’ C1’

T A

C G C1’

C1’


N

N

O

O

H

CH3 X6•T17

N

N

O

O dRib

CH3

N

NN

N

N

dRib

NO

OO

H

H H

N

N

O

O dRib

CH3

N

NN

N

N

dRib

NO

OO

H

H

H

C1 G2 T3 A4 C5 X6 C7 A8 T9 G10 C11

G22 C21 A20 T19 G18 T17 G16 T15 A14 C13 G12

dA-(N6)-ALII Duplex


H5’ H5’’

Selective H5” thymine deuteration

dA-(N6)-ALII Duplex

Lukin & de los Santos (2010) Nucleos. Nucleot. Nucleic Acids., 29, 562-573.

dA-(N6)-ALII Duplex


-9.5 -11.9 -203 C - A - G 0.8

-8.7 -11.0 -190 C - X - G

-10.5 -13.1 -219 T - A - G 1.2

-9.3 -11.2 -157 T - X - G

-12.7 -15.9 -273 C - A - C 2.5

-10.2 -12.7 -211 C - X - C

42.9

39.9

46.7

43.1

51.6

45.2

Tm (°C) Duplex Sequence

-72

-68

-78

-58

-98

-76

ΔH0

(Kcal/mol) ΔS0

(cal/mol °K) ΔG0

25°C (Kcal/mol)

ΔG037°C

(Kcal/mol) ΔΔG0

37°C (Kcal/mol)

dA-(N6)-ALII causes duplex destabilization


3.11

3.13

3.15

3.17

3.19

3.21

3.23

-1 4 -13.5 -13 -1 2.5 -12 -11.53.

123.

143.

143.

163.

183.

20-14.0 -13.5 -13.0 -12.5 -12.0

ln Ct10

00/T

m (K

-1)

CXC

CXG

TXG

The AL-II-dA lesion reduces the stability of the duplex

Duplex structure is perturbed at the lesion site. The AL-II moiety intercalates in the opposing strand

displacing the partner thymine in the major groove

In spite of these changes, the duplex adopts a single conformation that is stabilized mainly by hydrophobic

interactions at lesion site



dA-(N6)-AL-II adduct levels are elevated in GG-NER and transcription-coupled NER deficient cells, but

not in XPC cell lines lacking GG-NER.

In vitro, plasmids containing a single dA-(N6)-AL-II adduct are resistant to the early recognition and

incision steps of NER.

Additionally, XPC-RAD23B, the GG-NER damage sensor, failed to specifically bind to dA-(N6)-AL-II

adducts. Sidorenko et al (2012) Nucleic Acids Res. 40, 2494-2505

Recognition of bulky lesions by GG-NER seems less efficient than initially thought

Topology of duplex stabilizing lesions (AAF-N2-dG, 3ABA-N2-dG)

Stability threshold

Other factors

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Carlos de los Santos: TODAY’S SPEAKER - National … · 2012-05-02 · Carlos de los Santos:...

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