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 omas Lindahl Nobel Lecture, December 8, 2015

The Stability of DNA The Stability of DNA

O ! N ! C ! H ! N ! C ! Guanine Guanine H-C ! C ! C ! N ! N ! NH 2 ! O ! ! DNA lability DNA lability O ! P ! O ! CH 2 ! (especially (especially depurination depurination) ) ! O ! NH 2 ! O - ! ! H ! C ! N ! C ! Cytosine Cytosine H ! O ! C ! C ! O ! P ! O ! CH 2 ! H ! O ! N ! ! O ! C ! O - ! C ! H 3 C ! C ! N-H ! ! O ! H ! Thymine Thymine C ! C ! O ! P ! O ! H ! CH 2 ! N ! O ! O ! Sites susceptible to Sites susceptible to: : ! NH 2 ! O - ! ! N ! C ! Hydr Hydrolytic attack olytic attack C ! N ! O ! H ! Adenine Adenine H-C ! C ! C ! Oxidative damage Oxidative damage O ! P ! O ! N ! N ! H ! CH 2 ! ! O ! O - ! Alkylation, e.g. methylation Alkylation, e.g. methylation by by S -adenosylmethionine -adenosylmethionine O ! H ! ! Lindahl Nature 1993 3

O ! N ! C ! H ! N ! C ! H-C ! C ! C ! DNA lability DNA lability: : N ! N ! NH 2 ! O ! ! Deamination of Deamination of O ! P ! O ! CH 2 ! Cytosine Cytosine ! O ! NH 2 ! O - ! Hydrolytic Hydr olytic ! attack attack H ! C ! N ! C ! H ! O ! C ! C ! ! O ! P ! O ! CH 2 ! H ! O ! N ! ! O ! C ! O - ! C ! H 3 C ! C ! N-H ! ! O ! H ! C ! C ! O ! P ! O ! H ! CH 2 ! N ! O ! O ! ! NH 2 ! O - ! ! N ! C ! C ! N ! O ! H ! H-C ! C ! C ! O ! P ! O ! N ! N ! H ! O ! O - ! O ! H ! 4

Deamination of Cytosine Changes Coding Specificity Deamination of Cytosine Changes Coding Specificity deamination cytosine cytosine uracil uracil adenine adenine (nor (normally base pairs with guanine) mally base pairs with guanine) 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 3x10 9 9 bp genome of double-stranded DNA after 24h at 37°C) bp genome of double-stranded DNA after 24h at 37°C) 100% dsDNA Hydrolysis Hydr olysis 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-Hydroxyguanine (8-oxoG) 8-Hydr oxyguanine (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 oxidation products (M oducts (M 1 G, 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 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 C ! AP endonuclease AP endonuclease C ! phosphod phosphodiesterase iesterase C ! DNA polymerase DNA polymerase G ! C ! DNA l DNA ligase igase G ! C ! Lindahl Nature 1993 8

Reconstitution of Base Excision Repair Reconstitution of Base Excision Repair with Purified Human Pr with Purified Human Proteins oteins § 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 U G uracil-DNA glycosylase uracil-DNA glycosylase G AP endonuclease AP endonuclease G phosphodiesterase phosphod iesterase G polymerase polymerase C G ligase igase C G Kubota et al., EMBO J 1996 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 AID uracil-DNA glycosylase Neuberger & Rada, J. Exp. Med. 2007 11

O ! N ! C ! H ! DNA DNA lability lability: : N ! C ! Guanine Guanine H-C ! Oxidative damage Oxidative damage C ! C ! N ! N ! NH 2 ! O ! ! O ! P ! O ! CH 2 ! ! O ! NH 2 ! O - ! ! H ! C ! N ! C ! Cytosine Cytosine H ! O ! C ! C ! O ! P ! O ! CH 2 ! H ! O ! N ! ! O ! C ! O - ! C ! H 3 C ! C ! N-H ! ! O ! H ! Thymine Thymine C ! C ! O ! P ! O ! H ! CH 2 ! N ! O ! O ! ! NH 2 ! O - ! ! N ! C ! C ! N ! O ! H ! Adenine Adenine H-C ! C ! C ! Sites susceptible to O ! P ! O ! N ! N ! H ! CH 2 ! oxidative damage ! O ! O - ! O ! H ! Lindahl Nature 1993 12

Oxidative Damage to DNA: Oxidative Damage to DNA: Repair of Highly Mutagenic Lesions Repair of Highly Mutagenic Lesions 13

O ! N ! C ! H ! N ! C ! Guanine Guanine H-C ! C ! C ! DNA lability DNA lability: : N ! N ! NH 2 ! O ! ! Alkylation Alkylation O ! P ! O ! CH 2 ! ! O ! NH 2 ! O - ! ! H ! C ! N ! C ! H ! Cytosine Cytosine O ! C ! C ! O ! P ! O ! CH 2 ! H ! O ! N ! ! O ! C ! O - ! C ! H 3 C ! C ! N-H ! ! O ! H ! Thymine Thymine C ! C ! O ! P ! O ! H ! CH 2 ! N ! O ! O ! ! NH 2 ! O - ! ! N ! C ! C ! N ! O ! H ! Adenine Adenine H-C ! C ! C ! O ! P ! O ! N ! N ! H ! CH 2 ! ! O ! O - ! S –adenosylmethionine –adenosylmethionine O ! H ! Lindahl Nature 1993 14

Three Mechanisms that Repair Methylated DNA Bases Thr ee Mechanisms that Repair Methylated DNA Bases CH 3 NH 2 NH 2 NH 2 O H 3 C H 3 C N N N N N N N + + + N N O N N N N H 2 N N CH 3 DNA DNA DNA DNA O 6 -meG 3-meA 3-meA -meG 1-meA 1-meA 3-meC 3-meC AAG ! MGMT ! ABH2/3 ! + Fe + Fe 2+ 2+ + AP endonuclease + AP endonuclease + polymerase + polymerase + O 2 + O + DNA ligase + DNA ligase + α KG + KG + " CH CH 3 + " + " cys cys HCHO HCHO ee 3-meA 3-meA Fr Free + CO 2 + CO MGMT ! + succinate + succinate Base excision Base excision Direct damage r Dir ect damage reversal eversal repair epair 15

Oxidative Demethylation of 1-meA and 3-meC by AlkB Oxidative Demethylation of 1-meA and 3-meC by AlkB 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 16

The Intrinsic Fragility of DNA: The Intrinsic Fragility of DNA: Gr Group-specific Reagents Causing DNA Damage in Cells oup-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? ot? GRA GRAVEDIGGER VEDIGGER I' faith -- he will last you some eight year I' faith -- he will last you some eight year Hamlet Act 5: Or nine year: a tanner will last you nine year Or nine year: a tanner will last you nine year. . William Shakespeare HAMLET HAMLET Why he mor Why he more than another? e than another? GRAVEDIGGER GRA 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 of your wretched dead body etched dead body