Thomas A. Winters

Repair of Radiation-Induced DNA Double-Strand Breaks is Dependent upon Radiation Quality and the Structural Complexity of Double-Strand Breaks

Abstract Pastwa, E., Neumann, R. D., Mezhevaya, K. and Winters, T. A. Repair of Radiation-Induced DNA Double-Strand Breaks is Dependent upon Radiation Quality and the Structural Complexity of Double-Strand Breaks. Radiat. Res. 159, 251–261 (2003). Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co γ rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by γ rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of γ-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3′-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break

Abstract Datta, K., Jaruga, P., Dizdaroglu, M., Neumann, R. D. and Winters, T. A. Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break. Radiat. Res. 166, 767–781 (2006). Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted 125I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the 125I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the 125I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the ∼32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the 125I-decay site that may form via a charge migration mechanism.

Characterization of a complex 125I-induced DNA double-strand break: Implications for repair

Purpose: To examine the role of radiation-induced DNA double-strand break (DSB) structural organization in DSB repair, and characterize the structural features of 125I-induced DSBs that may impact the repair process. Methods: Plasmid DNA was linearized by sequence-specific targeting using an 125I-labeled triplex-forming oligonucleotide (TFO). Following isolation from agarose gels, base damage structures associated with the DSB ends in plasmids linearized by the 125I-TFO were characterized by probing with the E. coli DNA damage-specific endonuclease and DNA-glycosylases, endonuclease IV (endo IV), endonuclease III (endo III), and formamidopyrimidine-glycosylase (Fpg). Results: Plasmid DNA containing DSBs produced by the high-LET-like effects of 125I-TFO has been shown to support at least 2-fold lower end joining than γ-ray linearized plasmid, and this may be a consequence of the highly complex structure expected near an 125I-induced DSB end. Therefore, to determine if a high density of base damage exists proximal to the DSBs produced by 125I-TFOs, short fragments of DNA recovered from the DSB end of 125I-TFO-linearized plasmid were enzymatically probed. Base damage and AP site clustering was demonstrated within 3 bases downstream and 7 bases upstream of the targeted base. Furthermore, the pattern and extent of base damage varied depending upon the presence or absence of 2 M DMSO during irradiation. Conclusions: 125I-TFO-induced DSBs exhibit a high degree of base damage clustering proximal to the DSB end. At least 60% of the nucleotides within 10 bp of the 125I decay site are sensitive to cleavage by endo IV, endo III, or Fpg following damage accumulation in the presence of DMSO, whereas ⩾ 80% are sensitive in the absence of DMSO. The high degree of base damage clustering associated with the 125I-TFO-induced DSB end may be a major factor leading to its negligible in vitro repair by the non-homologous end-joining pathway (NHEJ).

Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures.

Abstract Datta, K., Weinfeld, M., Neumann, R. D. and Winters, T.A. Determination and Analysis of Site-Specific 125I Decay-Induced DNA Double-Strand Break End-Group Structures. Radiat. Res. 167, 152–166 (2007). End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cells ability to repair DSBs. The 3′-end groups of strand breaks caused by γ radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3′-phosphoglycolate and 3′-phosphate, with 5′-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3′-hydroxyl (3′-OH) and 3′-phosphate (3′-P) end groups and 5′-ends (≥42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3′-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

Coincident in vitro analysis of DNA-PK-dependent and -independent nonhomologous end joining

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA-PKcs to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA-PKcs appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA-PKcs protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.