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Ionizing radiation and double-strand break repair Kevin D. Brown, Ph.D. Electromagnetic Spectrum Ionizing radiation Gamma Rays 0.01 nm X-Rays 1 nm Ultra Violet 100 nm Visible Spectrum Infrared 400-700 nm 1 mm Radio Waves (Microwave, TV, Radio) 1 meter 1 km Sources of Ionizing Radiation Ionizing Radiation as a Genotoxin IR exposure can damage DNA due to both direct and indirect effects. Direct effects = DNA damage due to bombardment with γ-rays. Indirect effects = IR causes the formation of reactive species (e.g. H2O2, OH-) IR exposure results in a myriad of damage to bases Walker page 21 Deoxyribose damage Damaged bases can lead to destabilization of glycosidic bonds leading to abasic sites within the DNA • OH radicals can lead to destabilization of ribose ring structures Generation of abasic site Phosphodiester backbone damage (strand breaks) ss breaks result in damage that cannot be repaired via simple re-ligation (while 5ʼ PO4 groups are usually present, the 3ʼ end is not generally a simple OH group; rather phosphate and phosphoglycolate groups are present as well as damaged sugars and completely missing bases (nucleotide gaps) The exact mechanisms that give rise to ds breaks is unknown (two ss breaks?, different mechanism than ss breaks?) 1 Gy (100 Rad) of γ-rays results in 600-1000 ss breaks, 16-40 ds breaks and ~250 damaged bases (primarily thymidine) DSB repair - historical perspective Krasin and Hutchinson (1977) found that survival following IR exposure in E. coli was enhanced in bacteria containing replicated genomes. Pollard, Fluke and Kazanis (1981) observed that bacteria that carry multiple copies of the genome exhibit increased resistance to IR. These observations, as well as similar findings in yeast, suggest that alternate copies of the genome are used in repair mechanisms Homologous Recombination-based DSB repair MRN-dependent Rad51-dependent (RPA, Rad51, Rad52) Mitotic recombination Bacteria and yeast, homologous recombination DSB repair is the principal mechanism of DSB repair. In higher eucaryotes, non-homologous end joining (NHEJ) or single-strand annealing (SSA) are the primary mechanisms for DSB repair. Single-strand annealing 3ʼ Direct repeats IR 5ʼ to 3ʼ endonucleases Single Strand alignment 3ʼ Removal of Non-homologous 3ʼ ends DNA synthesis Ligation Molecules involved in SSA SSA occurs independent of Rad51 -- no strand invasion Because no homologous chromosome used in repair! However, Rad52 is required because there is strand annealing MRN is required for strand resection The mismatch repair proteins Msh2 and Msh3 as well as Rad1 and Rad10 are required for efficient SSA and appear to be needed to remove the non-homologous 3' tails from the annealed intermediate. Non-Homologous End Joining MRN, WRN, Artemis Molecules involved in NHEJ Ku70/Ku80 heterodimer: Avidly binds to DNA ends. The regulatory subunits of DNA-PKcs DNA-PKcs: Protein kinase, activity activated by free DNA ends. Relevant substrates are unknown. MRN complex, WRN helicase, Artemis endonuclease: Strand resection and end processing. XRCC4/Ligase IV complex: Required for ligation Repair factors are recruited to sites of damage Kim et al (2005) Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells. JCB, 170:341-347. DSB repair and genetic fidelity HR-based DSB repair: Because homologous chromosome is used, no loss of genetic information SSA: Loss of information that lies between sites of homology used in repair. NHEJ: Loss of genetic information due to strand resection. Because both SSA and NHEJ do not utilize homologous chromosomes for repair, it is possible that repair could result in translocation of genetic material. Surviving Fraction (%) DSB repair: It’s all about survival 100 10 1 0.1 0.01 0.001 0 NHF GM5849C (A-T) RKO LoVo HCT-116 2 5 10 IR Dose (Gy) 15 A single unrejoined DSB per cell per lethal event in yeast, suggesting that as little as 1 to 2.5% of the double strand breaks produced by ionizing radiation are never rejoined and become lethal. RPA H2AX MRN complex ATM ATR Chk1 Chk2 C-Abl The MRN complex localizes to the sites of DNA damage The proteins MRE11, Rad50, and Nbs-1 (XRS2 in yeast) form a complex termed MRN (MRX in yeast) Human and yeast MRE11 homologs have Mn-dependent nuclease activity in vitro. Predominantly, MRE11 displays 3’ - 5’ exonuclease activity and MRN is thought to act in strand resection during meiotic recombination and DSB repair. The pig pile at the DSB The pig pile is catalyzed by ATM-dependent phosphorylation reactions and binding of BRCT domain-containing proteins (53BP1, MDC1, BRCA1, NBS1 (the “N” of MRN)) Dysregulated coordination between lagging/leading strand synthesis RPA-bound Single stranded DNA The dance at the stalled fork 1) The kinase ATR binds to RPA coated ssDNA through its binding partner ATRIP 2) The 9-1-1 complex (Rad9/Hus1/Rad1), a PCNA-like clamp is loaded on the DNA at the stalled fork. The clamp loader is RFC where the largest of the subunits (RFC-1) is substituted with Rad17. 3) Rad9 is phosphorylated by ATR 4) BRCT domains on TopBP1 (topoisomerase-binding protein 1) associate with the phosphorylated sites on Rad9. 5) The completion of the 9-1-1/TopBP1/ATR complex fully activates ATR which then subsequently activates the kinase Chk1 ATM and ATR activate overlapping pathways that trigger cell cycle arrest, apoptosis, DNA repair and other DNA damage response (DDR) mechanisms