ROS, RNI, DNA Damage and Repair Signaling

ROS, RNI, DNA Damage

and Repair Signaling

Lynn Harrison, Ph.D.Department of Molecularand Cellular Physiology

Endogenous Cellular Factors That Damage DNA

• Replication errors– e.g. imbalance in the nucleotide pools– result in mismatch

• DNA instability– deamination of bases– depurination/depyrimidination of DNA

• loss of base

• Reactive oxygen and nitrogen species– DNA oxidation and deamination

Types of DNA Damage

• Base Damage

• Loss of bases - abasic or apurinic sites

• Strand breakage

• Protein-DNA cross-links

• DNA-DNA cross-links

Reactive oxygen species• Produced by normal cellular metabolism

• Mitochondria utilize ~85% O2 in cell and are a major source of ROS

• Damage DNA, protein and lipid.

• Some forms in cell are:– Hydrogen peroxide (H2O2)

– Superoxide radical (O2•-)

– Nitric oxide (•NO)– Hydroxyl radical (HO•)

• Fenton Reaction– Metal-catalyzed formation of HO• radicals. This

reaction can function in a redox cycle in which a transition metal ion transfers electrons from donors.

DNA(FeII) + H2O2 DNA(FeIII) + OH + OH-

• Hydroxyl radicals– Small modifications to bases, many types/base

– Single and double strand breaks

– Abasic sites (loss of base)

– Approximately 100 different types of damages identified

dR

Thymine

The Chemistry of Nitric OxideDictates its Physiological Activity

Oxidation

RNOS

Guanylate CyclaseCytochromes

C,O,N Radicals(Lipid Radicals)

O2 or O2-

Direct

Indirect

L-ArginineeNOSnNOSiNOS

NO

Nitrosation DNA Strand BreaksLipid Peroxidation

HydroxylationNitrosothiolsNitrosamines

NitrotyrosineNitroguanosine

Nitration

Metal Complexes/Alkyl Radicals

Nitrosamine-Mediated Alkylation of DNA Bases

Hydroxylation

N-Nitrosodimethylamine

O6Methylguanine Guanine

CH3N2+

+

CH2O + H2O

HN

N N

N

OCH3

H2N

N N

O

H3C

H3CN N

O

H2C

H3C

O H

HN

N N

N

O

H2N

P450

ArNH2 + N2O3 ArNHNO

ArNHNO + H+ ArN2+

+ H2O

ArN2+

+ H2O ArOH + N2 +H+

Nitrosative Deamination of DNA Bases by NO-Derived N2O3

ArNH2 represents DNA bases containing an exocyclic amino group: Cytosine, methylCytosine, Guanine or Adenine

Deamination of bases

BASE DEAMINATION PRODUCT

PAIRING AFTER

REPLICATION

MUTATION

C Uracil A G:C A:T

A Hypoxanthine C A:T G:C

G Xanthine No stable pair Block to replication

5MeC Thymine A G:C A:T

Biological Consequences of DNA Damage

• Block to DNA replication– e.g. thymine glycol, abasic site

• Mutagenesis– e.g. 8-oxoguanine pairs with A as well as C

Guanine

dR

• Deletions– Common with ionizing radiation– Due to strand breakage

• Chromosomal aberrations– Double strand breaks on different chromosomes

or chromatids

• Aberrant transcription– Breakage or abasic sites believed to result in

reduced transcription– Gaps in DNA result in deleted transcripts– Consequences are altered/ mutated proteins

Types of Repair

• Mismatch Repair– Repairs mismatches, these can be generated by replication

• Nucleotide excision repair– Repairs bulky lesions predominantly, also some small

oxidative lesions• Base excision repair

– Repairs small lesions e.g. oxidative damage and deaminations

• Non-homologous end-joining– Repairs double-strand breaks in all phases of the cell cycle

• Homologous recombination– Repairs double strand breaks in the S or early G2 phases of

the cell cycle

Summary

What activates the “alarm” signals after DNA damage

• Repair proteins that initiate the different pathways recognize lesions specifically.

• Believed to be constantly “patrolling” the DNA for damage, since damage is constantly occurring.

• If repair cannot handle the damage then the cell cycle checkpoints and possibly apoptosis are activated.

• Exception maybe the generation of a DSB where signaling is rapid. Activation may occur at a similar time as repair.

Initiation of signaling

• ATM and ATR are members of the PI-3-kinase-like family of kinases.• Much of the signaling is through protein phosphorylation

ROS, RNI

e.g. BER

Sustained ssDNA

RPA

Cell cycle checkpoint

Jeggo & LobrichRadiation Protection Dosimetry2007

Activation of ATR

ROS, RNI

Cell cycle checkpoint

Occurs in S phase due to replication blockOr when there is excessive ssDNA

Jeggo & LobrichRadiation Protection Dosimetry2007

ATR signaling due to RPA bound to ssDNA caused by stalled replication or blocking lesion

• MCM2-7– Replicative DNA helicase,

unwinds the DNA

• Cdc45– Essential for initiation and

elongation of DNA synthesis

• Pol , and – DNA polymerases

• PCNA– Proliferating cell nuclear

antigen

• RPA– Replication protein A, binds

to ssDNA Not generate ssDNA

DNA Repair 6, 953

RPA on SSDNA

ATR + ATRIP (P by ATR, essential for activity)

P

Rad9-Hus1-Rad1(Binds to TopBP1,

Required for activationof ATR kinase)

Rad17 (P by ATR, aids loading 911

binds to claspin)

Claspin(P by ATR

Needs Rad17)

Assembly on ssDNA

P

TopBP1(9-1-1 loads TopBP1

+ activates ATR)

P

After loading, Chk1 that is associated with the chromatin is phosphorylated

ATR + ATRIPP

Rad9-Hus1-Rad1

Rad17

P

TopBP1

Chk1P

• Claspin channels ATR to phosphorylate Chk1

• TopB1 assists ATR-ATRIP in phosphorylating numerous substrates including Chk1

ClaspinPP

ATR phosphorylates Chk1 to stop progression to mitosis

• DNA damage causes blockage of cell cycle

Chk1

Chk1

Chk1Chk1

Chk1

Chk1

Chk1 Chk1

Chk1

Chk1Chk1

Chk1

Progression S→M

Cdc25phosphorylase

P

cytoplasm

Wee1kinase

P

Adapted from Current Biology 16, 150

Results of Activation of ATR and Chk1DNA Repair 6, 953

Summary S phase damageDNA Repair 6, 953

Most DSBs are repaired by homologous recombination in S phase

Mre11/Rad50/Nbs1

RPA binding

Mutation Research 614, 95

Activation of ATM and DSB repair

ROS, RNI

Occurs when there is a double strand breakCan be cross-talk between the two kinases ATM and ATR

Jeggo & LobrichRadiation Protection Dosimetry2007

End-bindingand synapsis

Terminal processing

Ligation

Mammaliancells

Ku70, Ku80DNA PKcs

PNK 3’ P’aseArtemis

Fen1WRN, BLM

BRCA1Pol (Pol)

Prim1 (Pol complex)

DNA Ligase IVXRCC4

Adapted from Wilson et al 2003TIBS 28,62.

Damage/ repair foci• Occurs if the protein is retained at the site of damage• Can be produced with ionizing radiation either

targeted to the nucleus or by irradiating the whole cell• Can also be produced with a laser• After irradiation cells are fixed and

immunohistochemistry used to detect proteins• Or can use fluorescent tagged proteins and in the case

of the laser the proteins can be watched in real time as they move in the nucleus and redistribute from a diffuse appearance to high intensity foci

• Has been used to determine the order of proteins moving to the DNA

NHEJ proteins form foci only if there is a lot of damage

• Not possible to see Ku proteins well even under these conditions• Likely this is due to the fast on and off movement of the proteins• Note DNA-PKcs is visible within 10 seconds and only when Ku is present in

the cell

YFP-DNA-PKCs

Irradiation site

Pre 10” 60” 180”

YFP-DNA-PKCs

Pre 10” 60” 180”

Irradiation site

Cells contain Ku

Cells do notcontain Ku

Data from David Chen, UT Southwestern

ATM activation and NHEJ likely occur at the same time

• ATM is activated quickly and is found in foci• But the proteins involved in NHEJ are also activated

rapidly but not retained at the damage unless it is severe.

• Site of a DSB is marked by a chromatin modification called gamma H2AX

• H2AX is a variant of Histone 2A and it is phosphorylated and binds to the DNA near a DSB and spreads along the DNA up to a megabase pair flanking the break site

• Believed the resolution of the foci indicates repair

Activation of ATM

• ATM is a PI-3 kinase that phosphorylates proteins• Loss of this protein results in radiosensitization• It is required to block the cell cycle in G1, S and G2• Exists as an inactive dimer, chromatin changes believed

to cause activation and it autophosphorylates

Nature 421, 499

ATM activation does not require binding to a DSB

• Phosphorylation is detectable as soon as cells can be collected after irradiation and is maximal in 5 minutes after 0.5 Gy (~50% of protein is active)

• At 0.5 Gy there are only about 18 DSBs in the genome of the cell

• Can also be induced by a few restriction site cuts• Can be activated by hypotonic swelling of cells• Can be activated by chemicals known to alter

chromatin modifications and packaging e.g. trichostatin A

• Induction of breaks thought to cause relaxation of DNA structure and this is sensed by ATM

• ATM can also be activated by:– Retinoic acid (not

cause DNA damage)– MNNG when DNA

is not present– 15-deoxy-

delta(12,14)-prostaglandin J(2), which modifies SH groups

– REDOX activation?

Cold Spring Harbor Symposia on Quantitative Biology, vol LXX, 99-109Kitagawa & Kastan

Exo/endonucleaseNeeded for ATM

at DSB

Structural maintenance of chromosome family protein

Involved in blocking cell cycle in S phase

Breast cancer associated protein

1

1

2

3

Summary of signaling

• Phosphorylated ATM is needed for gamma H2AX modification. DNA PK from NHEJ may also do the phosphorylation

• Gamma H2AX required for repair foci formation

J. Cell Biol. 173,195

-53BP1 and MDC1 are mediators of ATM signaling

-They move to the DNA and are phosphorylated by ATM

-May be docking proteins for other signaling proteins

-No known activity associated but are required for ATM signaling

-BRCA1, MDC1 and 53BP1 needed for efficient autophosphorylation of ATM

Summary of proteins involved in signaling that are retained at damage

ATM signaling

ATR andHR

NHEJ

Checkpoint signaling

ATM signaling results in blockage in G1, intra S and G2/M

p53

Smc1Chk1+Chk2

Chk1Chk2

G1 block by p53

Cyclin A and E associates with Cyclin dependent kinase 2

G1 to S progression

p21

Chk2

Chk1 and Chk2

Chk1 and 2

Phosphorylation of Cdc25A (inactivates phosphatase)

↓ Cdk2-cyclin E

Prevents Cdc45 binding to origins of replication

Blocks replication and S phase

Chk2 also alters G2/MATM

MDC1

Chk2

↓ Cdc25 phosphorylase

↑ Phosphorylated Cdc2/cyclinB

Unable to progress to mitosis

Chk1

Summary of signaling

Reactive nitrogen intermediates result in stabilization of p53

Ref1 found to influence the transcriptional activity of p53

• Loss of Ref1 resulted in reduced induction of p21 and BAX

• Involved in transcriptional and hence p53 pro-apoptotic actions

ATM implicated in redox control in cells• ATM was found due to a human disease

– Ataxia Telangiectasia

• 1 per 40,000 live births, autosomal recessive• Cerebellar ataxia- staggering gait, severe muscular

uncoordination, progressive mental retardation• Ataxia – blood vessel dilation in the eyes• Increased cancer incidence• Defective immune system • Knockout mice have higher levels of oxidative stress,

found higher H2O2, without increase in catalase• Believe cells of CNS die due to this enhanced stress• Mechanism for ATM to control oxidative stress in cells is

not known

ATM also implicated in insulin response

• AT patients show glucose intolerance and insulin-resistance

• Insulin stimulation of cells seen to activate ATM and to release elF-4E allowing increased translation of specific transcripts

• ATM in vitro found to phosphorylate elF-4E binding protein 1

• ATM knock-out mice show delayed insulin secretion when posed with a glucose challenge

• Hence ATM maybe involved in insulin signaling• and metabolic function• Altering of metabolic function could be a cause of the

enhanced oxidative stress in the cells.

Bystander effect• Irradiation of one cell, triggers stress signals in

neighboring cells that were not hit by the radiation• Do not need to damage the DNA of the irradiated

cell• The bystander cells do get chromosomal

aberrations, mutation and it can cause transformation and cell death.

• Bystander cells may have DNA damage as signaling pathways are triggered as if DSB induction has occurred.

• Cytoplasmic irradiation and mitochondria as well as ROS and RNI have been implicated.