Repair of Radiation Damage

&

Dose Rate Effect

How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1, 22-33 (Oct 2001)

DNA Single Strand Repair

3 Pathways for Excision of damaged or inappropriate bases

• Base Excision Repair

• Mismatch Repair

• Nucleotide Excision Repair

Base Excision Repair

1) U is 1st removed by a glycosylase/DNA lyase

2) AP endonuclease then removes the sugar residue

3) Replacement w/correct nucleotide via DNA polymerase f3,

4) DNA ligase III-XRCC1-mediates ligation.

* Most common types of DNA damage induced by ionizing radiation are repaired through base excision repair.

U represents a putative single base mutation

Base Excision Repair

• If >1 nucleotide mutated

RFC/PCNA/DNA polymerase 8/ E synthesizes the damaged strand

FENI endonuclease removes the Overhanging flap

Ligase I DNA strands are sealed

Nucleotide Excision Repair

• Nucleotide excision repair (NER) removes bulky adducts in the DNA, such as pyrimidine dimers

Nucleotide Excision Repair

–(1) Damage recognition (XPC/HHRAD23B (R23, XPA, RPA, TFIIH and XPG)

-(2) Unwind (TFII), DNA incisions (XPG, ERCC1–XPF)

around the lesion, usually 24 - 32 nucleotides in length

-(3) Removal of the adducts

-(4) Repair synthesis to fill in the gap (TFII (XPB, XPD)-(5) DNA ligation (DNA Ligase)

How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1, 22-33 (October 2001)

e.g. Pyrimidine Dimer

DNA Bubble

• A mutation in NER genes does NOT lead to ionizing radiation sensitivity. – Increases sensitivity to UV-induced DNA damage & alkylating agents

that induce adducts.

Nucleotide Excision Repair (NER)

•Germline mutations in NER

genes lead to human DNA repair deficiency disorders such as xeroderma pigmentosum, in which patients are hypersensitive to UV light.

Mismatch Repair

• The mismatch repair pathway removes base-base mismatches in homologous recombination intermediates. Mutations in mismatch (MSH, MLH, and PSM) repair genes lead to microsatellite instability

• Precise Mechanism unknown

• Microsatellite Instability (small base insertions or deletions) and cancer, especially hereditary nonpolyposis colon cancer (HNPCC).

How nucleotide excision repair protects against cancer Errol C. Friedberg Nature Reviews Cancer 1, 22-33 (October 2001)

DNA Double Strand Repair (including HRR and NHEJ)

NHEJ is primary means of repairing dsDNA breaks

Defects lead to: IR sensitivity, chromosome aberrations

DNA Double Strand RepairNon Homologous End- Joining

(NHEJ)

1) Initiation (End recognition): - Binding of the Ku heterodimer to a dsDNA end

2) End Processing - Recruitment of DNA-dependent protein kinase catalytic

subunit, DNAPKcs Artemis activation - Artemis: Endonuclease dependent cleavage of overhangs

3) Synthesis: DNA polymerase

4) Bridging/Joining: XRCC4/DNA ligase IV/XLF complex

DNAPK: functions as a regulatory component of NHEJ, potentially facilitating and regulating the processing of the DNA ends. Recruits complex of three proteins, XRCC4, DNA ligase IV and XLF, which carry out the final rejoining step.

*Primary means of dsDNA break repair*Error prone* 2/2 in G1 w/no sister chromatid*Important for Ab diversity VDJ recombination

DNA Double Strand RepairHomologous Recombination (HR)

1) ATM/ATR (damage sensor) PO4’s H2AX

2) H2AX Recruits RAD51

3) RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52/BLM protects against exonucleolytic degradation.

4) Strand invasion (RAD54) D-loop Holliday jxn

5) Strand extension by DNA synthesis using sister chromatid template

6) Termination of the Holliday junction

Template = Error-free

*replication fork*

-H2AX recruits BRCA2

• NHEJ and HRR are not mutually exclusive, as both have been found to be active in the late S/G2

phase of the cell cycle (but rare)

DNA Double Strand Repair

NHEJ HR

Replication Associated Yes No

Phase of Cell Cycle G1 G2

BRCA Independent Dependent

Proliferation No effect Decreases

Sequence Loss Yes No (uses template)

ATM Independent Dependent

DNA RepairOther Processes

• Single-strand annealing (SSA) plays a transitional role between HRR and NHEJ, and results in loss of genetic information owing to the exonuclease degradation of the DNA ends.

• Cross-Link repair pathways used for DNA-DNA or DNA-protein cross-link repair are still under in vestigation.

PARP

Nature Reviews Cancer 10, 293-301 April 2010 OPINION:PARP inhibition: PARP1 and beyond Rouleau, Patel, Hendzel, Kaufmann & Poirier

- Synthetic Lethality = increases SSB w/in DNA

large amounsts of SSBs

collapse of replication forks during S phase

Accumulation of DSBs

*Central role in base excision repair so loss is Especially lethal in BRCA deficient cells as cant manage dsDNA Breaks

Syndromes that exhibit hypersentivity to ionizing

radiation…

AT

Cell-cycle checkpoints and cancer Michael B. Kastan and Jiri Bartek Nature 432, 316-323(18 November 2004) doi:10.1038/nature03097

ATR

SCIDSCID mice are immunodeficient due to defective DNA-PKcs Such cells are defective in nonhomologous recombination and are extremely radiosensitive.  ·SCID humans are also immune deficient and radiosensitive owing to a mutation in Artemis. Cells defective in Artemis are defective in nonohomologous recombination and are radiosensitive.

SCID & NBS NBS (Nijmegen Breakage Syndrome) is a very rare disorder that results in increased cancer incidence. Cells defective in NBS lack an S phase checkpoint and are radiosensitive.  ATLD (AT-Like Disorder) patients are clinically similar to AT patients except that their defect lies in the MRE7 7 gene. Cells from these patients are also sensitive to ionizing radiation.

Fanconi Anemia

FA (Fanconi Anemia) patients are characterized by their hypersensitivity to cross-linking agents. Although fibroblasts derived from these patients are not sensitive to ionizing radiation, tumors arising in these patients are hypersensitive. The reasons for this are currently unknown.

Operational Classifications of Radiation Damage

• Radiation damage to mammalian cells can be divided into…– Lethal damage – irreversible/irreparable and

leads irrevocable to cell death– Potentially lethal damage (PLD) – that which

can be modified by postirradiation conditions– Sublethal damage (SLD) – that which under

normal conditions can be repaired unless additional sublethal damage is added

Potentially Lethal Damage

The component of radiation damage that can be modified by manipulation of the postirradiation conditions is known as potentially lethal damage.  

Potentially Lethal Damage

Potentially lethal damage repair can occur if cells are prevented from dividing for 6 hours or more after irradiation; this is manifest as an increase in survival. This repair can be demonstrated in vitro by keeping cells in saline or plateau phase for 6 hours after irradiation and in vivo by delayed removal and assay of animal tumors or cells of normal tissues.

Potentially Lethal Damage

Sublethal Damage• The repair of sublethal damage is simply the repair of

double-strand breaks. • If a dose is split by a time interval, some double strand

breaks produced by the first dose are repaired before the second dose.

• Recall that double strand “lethal” damage may be formed by (1) single-track or (2) multiple-track damage.

• The component of cell killing resulting from single-track damage is the same whether the dose is a single exposure or fractionated. The same is not true of multiple-track damage.

Sublethal Damage

• For x-rays, split fractionation separated by 1 to 4 hours, results in a marked increase in cell survival because of the prompt repair of sublethal damage (multi-track damage).

• For neutrons, split fractionation offers little repair of sublethal damage (single track damage).

Sublethal Damage

Dose Rate Effect

Inverse Dose Rate

Dose Rate Effect

There is a dose-rate effect, such that at very low dose rates (<0.01 G/min) there is minimal checkpoint activation, probably due to minimal ATM activation.

At ~1 Gy/hr, only Late G2 checkpoint is triggered, leading to reassortment of cells at the G2/M interface (and resulting in the inverse dose rate effect). At higher dose rates, cell cycle progression is inhibited at all checkpoints

EXAMPLE

Dose Rate EffectEXAMPLE

To better illustrate the concept of inverse dose rate consider that…

• At lower dose rates cells may continue to progress through the cell cycle until they are arrested in G2, the most radiosensitive phase of

the cycle leading to greater cell kill. • By contrast, at higher dose rates, cells may

become "frozen" in a more radioresistant phase of the cycle leading to less cell kill.

Inverse Dose Rate

Brachytherapy

Brachytherapy

Brachytherapy

Brachytherapy

Radiolabeled Ig Therapy

Radiolabeled Ig Therapy

Radiolabeled Ig Therapy

Questions

• Which of the following is NOT a characteristic of DNA-protein kinase (DNA-PK)?

– A. DNA-PK consists of a catalytic subunit and two smaller accessory proteins, Ku70 (XRCC6) and Ku80 (XRCC5).

– B. It participates in the repair of DNA double strand breaks primarily through homologous recombination.

– C. Its loss in mice results in radiation sensitivity. – D. DNA-PK phosphorylates histone H2AX at sites of

double strand breaks. – E. It is a phosphatidyl-inositol-3-kinase.

• B DNA-PK is involved with non-homologous end-joining, not homologous recombination.

Answer

• Which of the following proteins is NOT involved in DNA repair?

– A. Artemis – B. RAD51 – C. DNA-PKcs – D. CDK4 – E. BRCA1

Questions

• D CDK4 is a cyclin dependent kinase that plays an important role in the progression of cells through G1 and into S phase. Artemis and DNA-PKcs play important roles in non-homologous end-joining of DNA double strand breaks, whereas RAD51 and BRCA1 are involved in the repair of double strand breaks through homologous recombination.

Answer

G1/S Phase Checkpoint

• SCID mice are often used in radiobiology research because they:

– A. are radioresistant – B. exhibit high levels of non-homologous end

joining – C. have efficient immune systems – D. are better able to repair radiation damage – E. are useful hosts for growing human tumor

xenografts

Questions

• E SCID mice are immune deficient, making them good hosts for growing xenografts of human tumors. SCID mice are deficient in DNA-PK and are therefore radiosensitive. Cells from these mice have low levels of non-homologous end-joining.

Answer

• Cells derived from individuals diagnosed with xeroderma pigmentosum are deficient in:

– A. nucleotide excision repair – B. methyl-guanine transferase – C. mismatch repair – D. base excision repair

Questions

• A People with xeroderma pigmentosum are deficient in one of the several proteins involved in nucleotide excision repair. They are therefore extremely sensitive to UV irradiation because they are unable to repair the pyrimidine dimers produced in DNA, but they are not sensitive to ionizing radiation.

Answer

• A mutation in NER genes does NOT lead to ionizing radiation sensitivity. – Increases sensitivity to UV-induced DNA damage & alkylating agents

that induce adducts.

Nucleotide Excision Repair (NER)

•Germline mutations in NER

genes lead to human DNA repair deficiency disorders such as xeroderma pigmentosum, in which patients are hypersensitive to UV light.

• Homologous recombinational repair of DNA double strand breaks is most likely to occur:

– A. in G0

– B. in G1

– C. in early S phase – D. in G2

– E. throughout the cell cycle

Questions

• D Homologous recombinational repair requires the presence of a homologous DNA template, and is therefore most likely to occur following DNA replication in G2

phase (when complete, sister chromatids are present in the cell).

Answer

• Which syndrome is caused by a deficiency in the repair-associated protein MRE11?

– A. Werner’s syndrome – B. Ataxia-Telangiectasia-like disorder – C. Xeroderma Pigmentosum – D. Bloom’s syndrome – E. Cocayne’s syndrome

Questions

• B A deficiency in MRE11 results in an ataxia telangiectasia-like disorder.

Answer

DNA Double Strand RepairHomologous Recombination (HR)

1) ATM/ATR (damage sensor) PO4’s H2AX

2) H2AX Recruits RAD51

3) RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52 protects against exonucleolytic degradation.

4) Strand invasion (RAD54) D-loop Holliday jxn

5) Strand extension by DNA synthesis using sister chromatid template

6) Termination of the Holliday junction

Template = Error-free

*replication fork*

-H2AX recruits BRCA2

• All the following statements are true concerning homologous recombinational repair of DNA double strand breaks, EXCEPT: – A. H2AX phosphorylation represents an important step in the

formation of repair foci. – B. The BLM protein serves to coat single stranded DNA

regions to prevent their degradation. – C. The MRN complex assists in producing single stranded

regions following the induction of a DNA double strand break. – D. RAD51 is a recombinase and forms a nucleoprotein

filament that facilitates strand invasion. – E. ATM is activated following irradiation by auto-

phosphorylation and conversion from an inactive dimer to the active monomer.

Questions

• B The RAD52/BLM protein is a helicase. RPA serves to coat single stranded DNA regions generated during homologous recombination to prevent their degradation.

Answer

DNA Double Strand RepairHomologous Recombination (HR)

1) ATM/ATR (damage sensor) PO4’s H2AX

2) H2AX Recruits RAD51

3) RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52 protects against exonucleolytic degradation.

4) Strand invasion (RAD54) D-loop Holliday jxn

5) Strand extension by DNA synthesis using sister chromatid template

6) Termination of the Holliday junction

Template = Error-free

*replication fork*

-H2AX recruits BRCA2

• All of the following statements about non-homologous end joining (NHEJ) are true, EXCEPT:

– A. Artemis is primarily responsible for ligating broken DNA ends.

– B. DNA ligase IV forms a tight complex with XRCC4.

– C. DNA-PKcs associates with Ku70/80 to form the DNA-PK holo-enzyme.

– D. The Ku heterodimer has a high affinity for DNA ends and forms a close-fitting asymmetrical ring that threads onto a free end of DNA.

– E. NHEJ is an error-prone process.

Questions

• A The main role for Artemis is to cleave (through its nuclease activity) any residual DNA loops or hairpins that form during non-homologous end-joining.

Answer

NHEJ HR

Replication Associated Yes No

Phase of Cell Cycle G1 G2

BRCA Independent Dependent

Proliferation No effect Decreases

Sequence Loss Yes No (uses template)

ATM Independent Dependent

• Which of the following is NOT a substrate for ATM?

– A. Ku70/80 (XRCC6/XRCC5) – B. BRCA1 – C. NBS1 – D. p53 (TP53) – E. CHK2 (CHEK2)

Questions

• A All of the proteins listed are substrates for ATM except for Ku70/80.

• Ku70/80 involved in NHEJ• NHEJ is ATM independent

Answer

DNA Double Strand RepairNon Homologous End- Joining

(NHEJ)1) Initiation (End recognition): - Binding of the Ku heterodimer to a dsDNA end

2) End Processing - Recruitment of DNA-dependent protein kinase catalytic

subunit, DNAPKcs Artemis activation - Artemis: Endonuclease dependent cleavage of overhangs

3) Synthesis: DNA polymerase

4) Bridging/Joining: XRCC4/DNA ligase IV/XLF complex

DNAPK: functions as a regulatory component of NHEJ, potentially facilitating and regulating the processing of the DNA ends. Recruits complex of three proteins, XRCC4, DNA ligase IV and XLF, which carry out the final rejoining step.

*error prone* 2/2 in G1 w/no sister chromatid*Important for Ab diversity VDJ recombination

• Which of the following statements is TRUE concerning DNA repair processes?

– A. Between 10-20% of the population is thought to be heterozygous for the types of mutations that are responsible for causing ataxia telangiectasia (AT).

– B. Non-homologous end-joining requires the involvement of a sister chromatid.

– C. Mutations in the genes that encode proteins involved in translesion DNA synthesis are typically present in people who develop hereditary non-polyposis colon cancer.

– D. The most common types of DNA damage induced by ionizing radiation are repaired through base excision repair.

Questions

• D The most common alterations produced in the DNA by radiation are base damages which are repaired by base excision repair, a repair process that is usually rapid and accurate.

• The proportion of the population that is heterozygous for the types of mutations that are found in people with AT – typically protein truncation mutations – is roughly 1-2%.

• Non-homologous end joining does not require a sister chromatid.

• Mutation of the genes involved with mismatch repair, primarily MSH2 and MLH1, are often present in people who develop hereditary non-polyposis colon cancer.

• Homologous recombination is a relatively error-free process.

Answer

• Normal tissue complications are most likely to be exhibited following conventional radiotherapy in patients suffering from:

– A. ataxia telangiectasia – B. systemic lupus erythematosus – C. Bloom's syndrome – D. xeroderma pigmentosum – E. Fanconi's anemia

Questions

• A Radiation injury would most likely occur in a person with ataxia telangiectasia. People with this syndrome are very sensitive to ionizing radiation due to the absence of functional ATM protein, which plays a central role in the repair of DNA double strand breaks and regulation of the cell cycle following irradiation.

Answer

• Repair of DNA double-strand breaks can be accomplished by which one of the following pathways?

– A. mismatch repair – B. non-homologous end joining – C. base excision repair – D. nucleotide excision repair – E. photoreactivation

Questions

• B Non-homologous end-joining represents the principal means by which human cells repair DNA double strand breaks. Mismatch repair is primarily responsible for correction of errors made during DNA replication.

• Base excision repair removes base damages.• Nucleotide excision repair mainly removes bulky

adducts from DNA such as UV-induced pyrimidine dimers and chemical adducts.

• Photoreactivation involves the action of DNA photolyase which is activated by long wavelength UV and visible light to split

Answer

DNA Double Strand Repair (including HRR and NHEJ)

NHEJ is primary means of repairing dsDNA breaks

Defects lead to: IR sensitivity, chromosome aberrations

RAD51 and BRCA2 function together: – A. as inhibitors of cyclin dependent kinases. – B. to phosphorylate H2AX and NBS1. – C. to enhance apoptosis by inhibiting p53

(TP53). – D. in the initial steps of homologous

recombination. – E. to play a central role in nucleotide excision

repair.

Questions

Answer

• D RAD51 and BRCA2 function DNA double strand breaks.

DNA Double Strand RepairHomologous Recombination (HR)

1) ATM/ATR (damage sensor) PO4’s H2AX

2) H2AX Recruits RAD51

3) RAD51 Initiates strand resection/exchange (MREII) & loading of RAD51 onto ssDNA, a process assisted by the BRCA2 protein. RAD52/BLM protects against exonucleolytic degradation.

4) Strand invasion (RAD54) D-loop Holliday jxn

5) Strand extension by DNA synthesis using sister chromatid template

6) Termination of the Holliday junction

Template = Error-free

*replication fork*

-H2AX recruits BRCA2