DNA Damage and DNA Repair

Thomas Begleytbegley@albany.edu

begley@email.unc.edu

mailto:tbegley@albany.edumailto:begley@email.unc.edu

DNADouble Helix

Purines

Pyrimidines

Nitrogen Bases of DNADNA

Endogenous DNA DamageEndogenouse Source Number of lesions

Hydrolysis Depurination 18,000 Depyrimidaination 600 Cytosine Deamination 100 5-methylcytosine deamination 10

Oxidation 8-oxoG ~1,000 - 2,000 Ring saturated pyrimidines ~2,000 Lipid Peroxidation products ~1,000

Nonenzymatic methylation by SAM 7-Methylguanine 6,000 3-Methyladenine 1,200 1-methyladenine and 3-methylcytosine ND

Nonenzymatic methylation by nitrosated polyamines abd peptides O6-methylguanine 20-100

Environmental Sources of DNA DamageReplication

problems

If DNA repair corrupted

Possible Consequences of DNA Damage

DNA DamageVia UV,

oxidation or methylation damage

Mutation

Cell Death

Nothing

Causes of Cancer and

Ageing

DNA Repair PathwaysComplexity

Nucleotide Excision RepairNucleotide Excision Repair

Mismatch RepairMismatch Repair

Base Excision RepairBase Excision Repair

+ Recombination based repair

Direct Repair

Mgm

t

Mgm

t

S-CH3SH

Direct Repair

Mgm

t

Mgm

t

S-CH3SH

Most Complex

http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.aspx

DNA Damaging Agents and Specific Types of DNA Damage

What is Ionizing Radiation?Ionizing radiation carries enough energy to ionize an atom or molecule (that is, to completely remove an electron from its orbit).

H O H O + + e-hv2 2

H2O + H+ + OH

DNA Repair and Mutagenesis By Errol C. Friedberg, Graham C. Walker, Wolfram Siede 1995

IR => ROS Damage

DNA Repair and Mutagenesis By Errol C. Friedberg, Graham C. Walker, Wolfram Siede 1995

UV DNA Damage

cis-syn and trans-syn diastereoisomers of cyclobutane dimers and the pyrimidine (6-4) pyrimidone adducts

Biol Chem, Vol. 275, Issue 16, 11678-11685, April 21, 2000

Formation of the Main UV-induced Thymine Dimeric Lesions within Isolated and Cellular DNA as Measured by High Performance Liquid Chromatography-Tandem Mass Spectrometry*

Thierry Douki, Magali Court, Sylvie Sauvaigo, Francette Odin, and Jean Cadet

Chemical CarcinogensDirect Acting Carcinogens

Alkylating agents Anticancer drugs (cyclophosphamide, chlorambucil, nitrosureas)

Procarcinogens that require metabolic activationPolycyclic and heterocyclic aromatic hydrocarbons

Benz(a)anthraceneBenzo(a)pyrene

Aromatic amines, amides, azo dyes2-Napthylamine2-Acetylaminofluorene

Natural plant and microbial productsAflatoxin B1Griseofulvin

OthersNitrosamineVinyl chlorideHeavy metals (Nickel & chromium)Insecticides, fungicidesPCBs

Alkylating Agents: Reactive Electrophiles

Simple alkylating agents found in tobacco smoke are an example

: most agents

: Sn1

: Sn2

: methyl radicals

N

N

O

NH2

NN

NNH

O

NH2dR

dR10% SS

0.1% 1.0 %

6.0 % 70%

32

6

3

78

C GN

NH

O

OCH3

NN

N

N

NH2

dR

dR

0.4 % 2.0 %

0.1%

20 %SS

10%

784

32 3T

A

Chemically Modified DNA

Carcinogenesis. 2004 Jun;25(6):1045-51.Alekseyev YO, Hamm ML, Essigmann JM.

Biochemistry 2002, 41, 9535-9544

Natalia Tretyakova,*,, Brock Matter, Roger Jones,| and Anthony Shallop

Types of DNA Repair Direct Repair (DR) Base excision repair (BER) Nucleotide excision repair (NER) Mismatch Repair (MMR) Recombinational repair (RR)

Direct Repair (DR)AlkB + Fe(II)

06-meG DNA Methyltransferase

06-meG DNA Methyltransferase

CH3Cys

06-meG DNA Methyltransferase

CH3Cys

N CH3

N CH3CH3O

O2 + -ketoglutarate

CO2 + succinate + HCHO

Direct Repair by MGMT

Mgmt

Mgmt

S-CH

3SH

NO N

N

O

NH

O

O

CH3

NH2

NO N

N

O

NH

O

ONH2

NO

NH

O

O

O

OCH3 CH3

NO

NH

O

O

O

OCH3

Direct Repair by AlkB like enzymes

ABH1, ABH4 - ABH8?

Base excision repair (BER)

Lots of flavors depending on the damage, initiating enzymes, and

downstream processing

AAG Substrates

N N

N+

NH

O

NH2*

CH3

N N+

N NH

O

NH2* CH3

N N+

N N

NH2

* CH3

N N

N N

NH

*

N N

N NH

O

*N N

NH

NH

O

NH2

O

*

N N

NH NH

O

NH2

CH2CH2

OH

*

N N

NH NH

O

NH2

CH2CH2

Cl

*

N N

N N

O

NH

*

7-MeG 3-MeG 3-MeA

A Hx 8-oxoG

G 7-HeG 7-CEG

BER: Initiated by a mono-functional glycosylase on alkylated DNA

(i.e. the AAG DNA Glycosylase)

Oxidative DNA Damage

OGG1 and MUTYHSubstrates

Removes ARemoves 8-oxoG

Nature 447, 941-950 (21 June 2007) |Sheila S. David1, Valerie L. O'Shea & Sucharita Kundu

BER: Initiated by a bi-functional glycosylase on oxidized DNA

(i.e. OGG DNA Glycosylases / AP Lyases)

Curr Biol. 1997 Sep 1;7(9):R576-9.Cunningham RP.

Three Distinct Ways to Address Oxidative Damage

Curr Biol. 1997 Sep 1;7(9):R576-9.Cunningham RP.

BER: Other enzymes and downstream decisions

DNA Repair (Amst). 2007 Mar 3; : 17337257

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification.

Karen H Almeida , Robert W Sobol

Nucleotide Excision Repair (NER)

http://www.youtube.com/watch?v=CASw429qBUU

http://www.youtube.com/watch?v=CASw429qBUU

Lesions Removed by NER

UV

cyclobutane dimer N2 BPDE-dG

Nucleotide Excision Repair

http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.aspx

xeroderma pigmentosum (XP) is caused by a defectin nucleotide excision repair mechanisms

XP is categorized in at least eight complementation groups according to the capacity of the body to repair DNA. These groups (i.e., genetic subtypes) are labeled A through

G, plus the XP variant:.

Wide range of symptomsblistering or freckling on minimum sun exposure

premature aging of skin, lips, eyes, mouth and tongue; with significant increased incidence of cancer in these same areas

blindness resulting from eye lesions or surgery for skin cancer close to the eyes progressive neurological complications including:

developmental disabilities mental retardation

The median age of onset of skin cancer is 8 years, nearly 50 years younger than in the general population

xeroderma pigmentosum (XP) is caused by a defectin nucleotide excision repair mechanisms

hypermutability after UV irradiation in cell cultures

propensity to cutaneous tumors after sun exposure (risk X 1000 to develop cancer on sun -exposed areas of the skin):

propensity to various solid tumors (mainly brain tumors, x 10 to 20 fold in comparison with general population )

DNA Repair and Mutagenesis By Errol C. Friedberg, Graham C. Walker, Wolfram Siede 1995

Mismatch Repair (MMR)http://www.youtube.com/watch?v=4XpQpDuLuhA

http://www.youtube.com/watch?v=4XpQpDuLuhA

Lesions Removed by MMR

J. Cell. Physiol. Vol.187, 2 Pages: 145-154

Mismatch Repair

Signaling by MMR and a MutatorPhenotype

J. Cell. Physiol. Vol.187, 2 Pages: 145-154

Recombinational Repair (RR)

Recombinational Repair1

Homologous recombination (HR)Non-homologous end joining (NHEJ)

IR Damage Results in Multiple Broken Chromosomes

http://en.wikipedia.org/wiki/DNA_repair

http://en.wikipedia.org/wiki/Image:Brokechromo.jpg

What happens if there is single strand break here?

double strand break => how do you repair?

General Mechanism of Homologous Recombination

Blunt duplex end converted into a 3 single stranded overhang coated with a filament of strand exchange proteins

Strand exchange filament invades homologous DNA, linking broken end to undamaged DNA duplex in 3 way junction (D-Loop)

D-loop converted to Holiday junction, followed by branch migration and resolution or invading strand is rejected

Occurs in late S and G2 of the cell cycle for Eukaryotes: why?

Non Homologous End Joining (NHEJ)

human

Interfaces with and participates in Recombination Repair. Protein complex 1 contains the A, B, C, E, F, G, L, M, and possibly I subunits. Protein complex 2 contains FANCD2-Ub, BRCA2, and possibly FANCJ. During S phase, when a replication fork encounters a DNA cross-link, the FA complex (complex 1) is activated. This activation leads to the monoubiquitinaiton of FANCD2, which is then targeted to chromatin containing the cross-link. FANCD2-Ub then interacts with BRCA2 in complex 2, leading to repair of the cross-link possibly through HR and TLS.

Fanconi Anemia Pathway

Chromosome breakage in Fanconi Anemia cells

http://www.rndsystems.com/mini_review_detail_objectname_MR03_DNADamageResponse.aspx

http://www.genesdev.org/content/vol19/issue24/images/large/2925fig1.jpeg

Endogenous and Environmental Sources of DNA Damage

Sensing DNA Damage

http://www.nature.com/nature/journal/v408/n6811/full/408433a0.html

Which repair pathway is described in the following movie?

http://www.youtube.com/watch?v=CcTayxEblio&feature=related

http://www.youtube.com/watch?v=CcTayxEblio&feature=related

Endogenous DNA DamagePossible Consequences of DNA DamageDNA Repair PathwaysIR => ROS DamageUV DNA DamageAlkylating Agents: Reactive ElectrophilesChemically Modified DNATypes of DNA RepairDirect Repair (DR)Direct Repair by MGMTDirect Repair by AlkB like enzymesBase excision repair (BER)BER: Initiated by a mono-functional glycosylase on alkylated DNA(i.e. the AAG DNA Glycosylase)BER: Initiated by a bi-functional glycosylase on oxidized DNA(i.e. OGG DNA Glycosylases / AP Lyases)Three Distinct Ways to Address Oxidative DamageNucleotide Excision Repair (NER)Mismatch Repair (MMR)Signaling by MMR and a Mutator PhenotypeRecombinational Repair (RR)Non Homologous End Joining (NHEJ)Sensing DNA Damage