Radiation Oncology/Radiobiology/DNA Repair

Repair of Radiation-Induced DNA Damage

Single Strand Damage Repair

• Single strand breaks are common occurrence; 1 Gy of radiation induces ~1000 SSB per cell
• Given this, cells have efficient machinery to repair SSB
• However, single strand breaks are also important to double strand break formation

1. Ionizing radiation often occurs in clusters, and some SSB will be created in proximity to areas with base damage. In these, the temporary strand nick performed during BER can combine with the near SSB to form a DSB
2. If SSB happens in a replication fork during S-phase, the fork collapses and there is a single-end double-strand break. These cannot be repaired by NHEJ (since there are not two ds breaks), and must be attempted to be repaired by HR. This may be a problem in tumors with HR deficiencies (such as BRCA1 and BRCA2 mutations), and PARP inhibition seems particularly effective in these settings

Base Excision Repair (BER)

• Major pathway responsible for repair of hydrolytic and oxidative base damage (e.g. Uracil, 8OH-guanine, abasic sites)
• Damage is recognized by Poly (ADP-ribose) polymerase (PARP)
• Depending on the number of bases damaged, BER proceeds through either Short Patch or Long Patch pathway
  • Short Patch BER
    • DNA glycosylase excises the base leading to AP Site (apurinic site). There are different glycosylases which recognize specific bases
    • AP endonuclease (APE-1) removes the sugar residue
    • DNA polymerase β replaces the correct nucleotide
    • DNA ligase III and XRCC1 complex ligate the free DNA strands together
  • Long Patch BER
    • DNA glycosylase excises the base leading to AP Site (apurinic site). There are different glycosylases which recognize specific bases
    • AP endonuclease (APE-1/AP Lyase) removes the sugar residues
    • PCNA (Proliferating cell nuclear antigen) serves as an adapter for proteins involved in the subsequent steps
    • DNA polymerase δ/ε and PCNA complex duplicate the correct nucleotide sequence, duplicating it even under the undamaged strand
    • Flap endonuclease 1 (FEN1) and PCNA complex snips off normal overhang of the prior undamaged strand
    • DNA ligase I and PCNA complex ligate the free DNA strands together
• Defects generally do not increase radiation sensitivity, but lead to increased mutation rate. Defect in XRCC1 does increase radiosensitivity (1.7X), but this may be due to its role in SSB repair
• Inhibition of PARP and XRCC1 is efficient in HR-deficient tumors
• Hereditary Syndromes:
  • No human diseases resulting from BER mutation have thus far been identified

Nucleotide Excision Repair (NER)

• Major pathway responsible for repair of bulky DNA adducts (e.g. pyrimidine dimers); as such, it is not critically important for radiation oncology
• Overview of the steps involved (see more at wikipedia
  • Incision in DNA surrounding the damage; this is typically a bracket 24 - 32 bp long
  • Removal of the entire region containing the adducts
  • Repair synthesis of DNA
  • DNA ligation
• Proteins involved include XPA,XPB, XPC,XPD,XPE,XPF, and XPG (linked to Xeroderma pigmentosum patients) and CSA (ERCC8) and CSB (ERCC6) (linked to Cockayne syndrome)
• Defects generally do not increase radiation sensitivity, but lead to increased sensitivity to UV damage and to anticancer agents that induce bulky addects (e.g. alkylating agents)
• Hereditary Syndromes:
  • Xeroderma Pigmentosum
  • Cockayne Syndrome

Mismatch Repair

• Pathway responsible for removes base-base mismatches and small insertion/ deletion mismatches
• Overview of the steps involved
  • Initiation: MSH2, MSH3, and MSH6 surround DNA; MLH1 and PSM2
  • Excision and resynthesis: Polymerase δ/ε and PCNA complex
  • DNA ligation with DNA ligase I
• Defects generally do not increase radiation sensitivity, but lead to increased mutation rate
• Mutations in MLH, MSH, or PSM families result in microsatellite instability (small base insertions or deletions) and cancer (e.g. sporadic endometrial cancer)
• Hereditary Syndromes:
  • Hereditary Non-Polyposis Colon Cancer (HNPCC).

DNA Damage Bypass

• Occurs when a moving replication fork encounters damage in the template strand
• Important component of cellular response to DNA damage, and contributes similar survival as NER and HR
• Rad 18 binds to DNA, Rad 6 ubiquitylates PCNA
• PCNA may determine if a recombinatorial bypass (error-free) or translesion bypass (error-prone) will take place. Not well understood
• SRS2 is a helicase, which blocks further homologous recombination
• Recombination bypass proteins include Rad5, Ubc13, Mms2
• Translesion bypass proteins include DNA polymerase ζ and η
• Please see Rosewell Park lecture for more information

DNA Alkylation

MGMT

• MGMT (O⁶-methylguanine-DNA methyltransferase) gene is located on chromosome 10q26
• It encodes DNA-repair proteins that removes alkyl groups from O⁶ position on guaning
• Repair of DNA by MGMT consumes the protein, which must be replaced by the cell
• O⁶-methylguanine lesions are induced by chemotherapy alkylating agents, and if left unrepaired, trigger cytotoxicity and apoptosis
• Epigenetic silencing of MGMT gene by promoter methylation results in loss of MGMT expression and reduced DNA repair. Please see the Glioblastoma chapter for more information

Double Strand Damage Repair

• There are two main pathways for repair of DSBs
  • Nonhomologous end-joining (NHEJ): primarily active during G0, G1, early S when sister chromatid not present, though also active in G2/M. Joins two double strand breaks. Error-prone
  • Homologous recombination repair (HRR): during G2/M when sister chromatid is present. Copies information from the undamaged chromosome. Error-free
• The role of single-strand annealing as a repair pathway for DSB has not been well clarified, but seems to involve homologous recombination as well

Nonhomologous End-Joining (NHEJ)

• Joins two DSB ends together, without requiring a template
• Low fidelity, with frequent deletions or insertions at the break site. While this may increase mutations, it allows the cell to survive. Also used for immunoglobulin V(D)J recombination
• Starts with Ku70/Ku80 heterodimer binding to free DNA ends, and acting as a scaffold
• It then recruits DNA-PKcs (PRKDC), which acts as a bridge between the two free DNA ends. It also phosphorylates a number of target proteins involved in DNA repair and cell cycle control
• DNA-PKcs complexes with Artemis, which is an endonuclease to process the DNA ends
• Several other enzymes, including nucleases and polymerases, then fill in or remove single-strand overhangs. This process can result in insertions or deletions
• Final step is ligation of the two ends together by ligase IV, assisted by XRCC4 and XLF
• Hypersensitivity to ionizing radiation
• Hereditary syndromes:
  • DNA-PKcs, Artemis, XLF: Radiosensitive Severe Combined Immunodeficiency (RS-SCID) (PMID 19075392)
  • LIG4 (Ligase IV gene): LIG4 syndrome, similar to SCID

Homologous Recombination (HR)

• Uses homologous DNA (replicated DNA) as a template to repair damage
• May take up to 6 hours or more to complete
• Starts with creating single strand overhang regions around each side of the break by resecting part of the 5' -> 3' strand, with MRN playing a major role
• MRN complex (MRE11-RAD50-NBS1) holds the broken ends of DNA, such that MRE11 attaches to the broken DNA ends, while RAD50 bridges the break, and NBS1 activates ATM to start the repair
• BRCA1 is recruited by H2AX to regulate the activity of the MRN complex
• Replication protein A (RPA) coats each single strand overhang as scaffold
• RAD51 complex attaches around the ssDNA to create a nucleoprotine filament and searches for the homologue strand. There are multiple versions of RAD51 that participate in this process (e.g. RAD51B, RAD51C, RAD51D, XRCC2, XRCC3)
• BRCA2 facilitates loading of RAD51 onto the RPA-covered single strand overhang
• FANC-D2 regulates function of BRCA2, and is also involved in S-phase checkpoint
• RAD52 and RAD54 are supporting proteins to RAD51 in this process
• Helicases BLM, TopIIIA, WRN, and others unwind the homologous DNA double strand; RAD54 may also play some role in this process
• The identity of polymerases and ligases involved in copying the damaged strand based on the homologous template is not known, although Ligase I is probably involved
• MMS4 and MUS81 resolve the Holiday junction crossovers
• Hereditary Syndromes:
  • MRE11:Ataxia-Telangiectasia-Like Disorder (ATLD)
  • NBS1: Nijmegen Breakage Syndrome (NBS)
  • RAD50: Nijmegen Breakage Syndrome-Like Disorder (NBSLD)
  • BLM: Bloom Syndrome
  • WRN: Werner syndrome
  • FANC-D2: Fanconi Anemia (FA)
  • ATM: Ataxia-Telangiectasia
  • ATR: ATR-Seckel syndrome

Single Strand Annealing (SSA)

• A transitional process between NHEJ and HR
• One strand on each side of the break is digested away, until a region of homology is found between the remaining two strands
• The two single strands are annealed at that point
• The complementary strands that were digested are reconstituted
• Results in loss of genetic information
• Please see this page for more detail and pictures

NHEJ vs HR

• NHEJ happens along in all phases of the cell cycle
• HR is specific only to dividing cells, since it occurs in S and G2 phase
• In G1 phase, double strand DNA damage is repaired with NHEJ, while in S-G2 phases, both processes are active and compete against each other
• Therefore, in slowly dividing or non-dividing tissues (e.g. spinal cord or stromal tissues), primary repair process is NHEJ
• There is some evidence that MRN is active in both processes, and that it may regulate the choice between NHEJ and HR in dividing cells
• If one of the components is defective, it leads to various genomic instability disorders and increased malignancy

Cross-Link Repair

• Genes and pathways used for DNA-DNA and DNA-protein cross-links are under investigation
• The pathway depends on homologous recombination

DNA Repair Literature

• Saarland (Germany); 2008 PMID 18805648 -- "DNA Double-Strand Break Rejoining in Complex Normal Tissues." (Rube Ce, Int J Radiat Oncol Biol Phys. 2008 Sep 19. [Epub ahead of print])
  • TBI in mice model. Formation and rejoining of DSBs analyzed via gammaH2AX in both early responding (small intestine) and late responding (lung, brain, heart, kidney) tissues
  • Outcome: Linear dose correlation of gammaH2AX to dose. Various normal tissues exhibited identical kinetics of gammaH2AX foc loss, despite different clinical radiation response
  • Conclusion: Identical kinetics of DSB rejoining in various organs suggest that tissue specifice difference in radiation response are independent of DSB rejoining

• Oxford; 2008 PMID 18571480 -- "Interplay of two major repair pathways in the processing of complex double-strand DNA breaks." (Dobbs TA, DNA Repair (Amst). 2008 Aug 2;7(8):1372-83. Epub 2008 Jun 20.)
  • Evaluation of base excision repair and DSB ligation
  • Conclusion: Reduced efficiency of repair of complex DSBs

• London Research Institute; 2008 PMID 18596698 -- "Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity." (Jazayeri A, EMBO J. 2008 Jul 3. [Epub ahead of print])

• Wayne State; 2007 PMID 17967316 -- "Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity." (Krueger SA, Int J Radiat Oncol Biol Phys. 2007 Nov 15;69(4):1262-71.)
  • Role of ATM and downstream G2 cell-cycle checkpoint in low-dose hyper-radiosensitivity

• Harvard; 2007 PMID 17525332 -- "ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage." (Matsuoka S, Science. 2007 May 25;316(5828):1160-6.)
  • Proteomic analysis of ATM/ATR phosphorylated proteins
  • >900 regulated sites found on >700 proteins, many involved in DNA damage response

ERCC1

• Impacts DNA damage from lesions caused by UV light, exogenous chemicals that induce bulky DNA adducts (eg cisplatin)
• 1/16 genes in nucleotide excision repair complex
• Forms heterodimer with XPF, which excises 5' DNA strand relative to site of damage. This is final step of the excision process
• PMID 16957145 -- "DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy." (Olaussen, N Engl J Med. 2006 Sep 7;355(10):983-91.)
  • Prognostic for benefit of chemo in resected NSCLC

Summary of Damage - Repair

• Thymine glycol - BER
• 8-OH-guanine - BER
• Urea/uracil - BER
• Bulky adducts, pyrimidine dimers - NER
• Nucleotide error - Mismatch repair
• DNA double strand break - NHEJ or HR

Review

• 2007 PMID 17512070 -- "DNA damage-induced signalling in ataxia-telangiectasia and related syndromes." (Lavin MF, Radiother Oncol. 2007 Jun;83(3):231-7. Epub 2007 May 23.)

• 2007 PMID 18066093 -- "DNA double-strand break repair and development." (Phillips ER, Oncogene. 2007 Dec 10;26(56):7799-808.)

• 2002 PMID 12016139 -- "Sensing and repairing DNA double-strand breaks." (Jackson SP, Carcinogenesis. 2002 May;23(5):687-96.)

• 1998 PMID 9806612 -- "Monitoring and signaling of radiation-induced damage in mammalian cells." (Szumiel I, Radiat Res. 1998 Nov;150(5 Suppl):S92-101.)