The Intrinsic Fragility of DNA The Intrinsic Fragility of DNA Tomas - - PowerPoint PPT Presentation

The Intrinsic Fragility of DNA The Intrinsic Fragility of DNA

Tomas Lindahl

• mas Lindahl

Nobel Lecture, December 8, 2015

The Stability of DNA The Stability of DNA

Sites susceptible to Sites susceptible to: :

Hydr Hydrolytic attack

• lytic attack

Oxidative damage Oxidative damage Alkylation, e.g. methylation Alkylation, e.g. methylation by by S-adenosylmethionine

• adenosylmethionine

!

O! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C! O! H! NH2 !

!

C! C! C! H! N! N! C! O! C ! C! C! C! H3C! ! N! N-H! C! O! P! O! O! CH2! ! O! O -! P! O! O! CH2! ! O -! O! H! P! O! O! CH2! ! O -! O! H! O! H! P! O! O! CH2! ! O -! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C!

DNA DNA lability lability

(especially (especially depurination depurination) )

Guanine Guanine Cytosine Cytosine Adenine Adenine Thymine Thymine

Lindahl Nature 1993

3

Hydr Hydrolytic

• lytic

attack attack

!

O! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C! O! H! NH2 !

!

C! C! C! H! N! N! C! O! C ! C! C! C! H3C! ! N! N-H! C! O! P! O! O! CH2! ! O! O -! P! O! O! CH2! ! O -! O! H! P! O! O! CH2! ! O -! O! H! O! H! P! O! O! O -! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C!

DNA DNA lability lability: :

Deamination of Deamination of Cytosine Cytosine

4

Deamination of Cytosine Changes Coding Specificity Deamination of Cytosine Changes Coding Specificity

deamination cytosine cytosine

(nor (normally base pairs with guanine) mally base pairs with guanine)

uracil uracil adenine adenine

5

Spontaneous DNA lesions in a Mammalian Cell Spontaneous DNA lesions in a Mammalian Cell

(number of alter (number of altered nucleotides in a 3x10 ed nucleotides in a 3x109

9 bp genome of double-stranded DNA after 24h at 37°C)

bp genome of double-stranded DNA after 24h at 37°C) Hydr Hydrolysis

• lysis

Depurination Depurination 9000 9000 Depyrimidination Depyrimidination 300 300 Cytosine deamination Cytosine deamination 50 50 5-Methylcytosine deamination 5-Methylcytosine deamination 5 5

Oxidation Oxidation

8-Hydr 8-Hydroxyguanine (8-oxoG)

• xyguanine (8-oxoG)

500-1000 500-1000 Ring saturated pyrimidines (thymine glycol, cytosine hydrates) Ring saturated pyrimidines (thymine glycol, cytosine hydrates) 1000 1000 Lipid per Lipid peroxidation pr

• xidation products (M
• ducts (M1G, etheno-A, etheno-C)

G, etheno-A, etheno-C) 1000 1000

Non-enzymatic methylation by S-adenosylmethionine Non-enzymatic methylation by S-adenosylmethionine

7-Methylguanine 7-Methylguanine 3000 3000 3-Methyladenine 3-Methyladenine 600 600 1-Methyladenine/3-Methylcytosine 1-Methyladenine/3-Methylcytosine 10-20 10-20

100% dsDNA

6

The high rate of DNA decay implies that The high rate of DNA decay implies that repair mechanisms must exist epair mechanisms must exist and ar and are continually active under physiological conditions e continually active under physiological conditions

7

Repair of Abasic Sites in DNA Repair of Abasic Sites in DNA

Lindahl Nature 1993

C! C! G! C! C! C! G!

AP endonuclease AP endonuclease phosphod phosphodiesterase iesterase DNA polymerase DNA polymerase DNA l DNA ligase igase

8

Reconstitution of Base Excision Repair Reconstitution of Base Excision Repair with Purified Human Pr with Purified Human Proteins

• teins

uracil-DNA glycosylase uracil-DNA glycosylase AP endonuclease AP endonuclease phosphod phosphodiesterase iesterase polymerase polymerase ligase igase Kubota et al., EMBO J 1996

U G G G G C G C G

§ DNA glycosylase r DNA glycosylase removes a damaged base, to cr emoves a damaged base, to create an AP site eate an AP site § The first such enzyme discover The first such enzyme discovered was uracil-DNA glycosylase ed was uracil-DNA glycosylase

9

Model for Base Excision Repair in Human Cells Model for Base Excision Repair in Human Cells

Mol, Pankh, Putman, Lo & Tainer Ann. Rev. Biophys. Biomol. Struct. 1999

10

When DNA Damage is a Good Thing: When DNA Damage is a Good Thing:

Generation of Antibody Diversity by Somatic Hyper Generation of Antibody Diversity by Somatic Hypermutation mutation

uracil-DNA glycosylase

AID

Neuberger & Rada, J. Exp. Med. 2007

11

Sites susceptible to

• xidative damage

O! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C! O! H! NH2 !

!

C! C! C! H! N! N! C! O! C ! C! C! C! H3C! ! N! N-H! C! O! P! O! O! CH2! ! O! O -! P! O! O! CH2! ! O -! O! H! P! O! O! CH2! ! O -! O! H! O! H! P! O! O! CH2! ! O -! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C!

DNA DNA lability lability: :

Oxidative damage Oxidative damage

Guanine Guanine Cytosine Cytosine Adenine Adenine Thymine Thymine

Lindahl Nature 1993

12

Oxidative Damage to DNA: Oxidative Damage to DNA:

Repair of Highly Mutagenic Lesions Repair of Highly Mutagenic Lesions

13

O! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C! O! H! NH2 !

!

C! C! C! H! N! N! C! O! C ! C! C! C! H3C! ! N! N-H! C! O! P! O! O! CH2! ! O! O -! P! O! O! CH2! ! O -! O! H! P! O! O! CH2! ! O -! O! H! O! H! P! O! O! CH2! ! O -! O! O! H! C! NH2 !

!

N! C! C! N! H-C! N! N! H! C!

DNA DNA lability lability: :

Alkylation Alkylation

Guanine Guanine Cytosine Cytosine Adenine Adenine Thymine Thymine

Lindahl Nature 1993

S –adenosylmethionine –adenosylmethionine

14

Thr Three Mechanisms that Repair Methylated DNA Bases ee Mechanisms that Repair Methylated DNA Bases

Dir Direct damage r ect damage reversal eversal 3-meA 3-meA

NH2

CH3

DNA N N N N

+ + AP endonuclease + AP endonuclease + polymerase + polymerase + DNA ligase + DNA ligase Fr Free ee 3-meA

3-meA

+ "

Base excision Base excision repair epair AAG! 1-meA 1-meA

DNA N N N N NH2

+ H3C

N N NH2

+

O

H3C

DNA

3-meC 3-meC + Fe + Fe2+

2+

+ O + O2 + + αKG KG

HCHO HCHO

+ CO + CO2 + succinate + succinate

+ "

ABH2/3! O6-meG

• meG

H2N DNA N N N N O

CH3

MGMT!

CH CH3 cys cys

+ "

MGMT!

15

The molecular mechanism employed for the demethylation of DNA The molecular mechanism employed for the demethylation of DNA is also used to demethylate histones is also used to demethylate histones

Oxidative Demethylation of 1-meA and 3-meC by AlkB Oxidative Demethylation of 1-meA and 3-meC by AlkB

16

The Intrinsic Fragility of DNA: The Intrinsic Fragility of DNA:

Gr Group-specific Reagents Causing DNA Damage in Cells

• up-specific Reagents Causing DNA Damage in Cells

Water (55 M in cells!) ater (55 M in cells!) Reactive oxygen Reactive oxygen S-adenosylmethionine (SAM) S-adenosylmethionine (SAM) Small r Small reactive molecules – e.g. for eactive molecules – e.g. formaldehyde maldehyde

Ø The d The diversity of lesions r iversity of lesions requir equires a d es a diversity of r iversity of repair enzymes epair enzymes Ø Many DNA lesions, and t Many DNA lesions, and the corr he correspond esponding r ing repair systems, may epair systems, may remain to be d emain to be discover iscovered ed

17

HAMLET HAMLET How long will a man lie i' the earth er How long will a man lie i' the earth ere he r e he rot?

• t?

GRA GRAVEDIGGER VEDIGGER I' faith -- he will last you some eight year I' faith -- he will last you some eight year Or nine year: a tanner will last you nine year Or nine year: a tanner will last you nine year. . HAMLET HAMLET Why he mor Why he more than another? e than another? GRA GRAVEDIGGER VEDIGGER Why Why, sir , sir, his hide is so tanned with his trade, that , his hide is so tanned with his trade, that He will keep out water a gr He will keep out water a great while; and your eat while; and your Water is a sor ater is a sore decayer e decayer of your wr

• f your wretched dead body

etched dead body

Hamlet Act 5: William Shakespeare