Gene targeted DNA double-strand break induction by 125l-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells

Mezhevaya, Katherina; Winters, Thomas A.; Neumann, Ronald D.

doi:10.1093/nar/27.21.4282

Cited by 19 publications

(11 citation statements)

References 59 publications

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“…tions described here resembles closely the one obtained previously in vivo by transfection of a 125 I-TFO-linearized plasmid in mammalian cells (40). This indicates that our in vitro system yields reliable results.…”

Section: End Joining Of 125 I-induced Double-strand Breakssupporting

confidence: 88%

“…Still, we do not know at present whether the reduced NHEJ efficiency for 125 I-TFO-induced DSB reflects the in vivo situation or simply is due to the lack in our extracts of some components necessary to remove damaged DNA moieties prior to NHEJ. To clarify this issue, transfection experiments similar to the ones described previously (40) would have to be performed to compare the joining capacity of 125 I-TFO-and RE-linearized plasmids in vivo. It is also worth mentioning that 125 I-TFO-induced DSB reduce ccc product formation to a greater degree than dimer formation in comparison with RE-induced DSB.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Odersky

Panyutin²,

Panyutin³

et al. 2002

Journal of Biological Chemistry

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In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of doublestrand break (DSB) repair. Rejoining of DSB produced by decay of 125 I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the 125 I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin 125 I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the 125 I-TFO within its target site. We show that NHEJ of 125 I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after 125 I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.Mammalian genomes constantly suffer a variety of types of damage, of which double-strand breaks (DSB) 1 are considered the most dangerous. DSB may arise spontaneously in the cell or may be induced by exogenous agents, such as ionizing radiation. The estimation that mammalian cells suffer at least 10 spontaneous DSB/day suggests that efficient repair of DSB is critical for cell survival (1). Failure to do so can result in deleterious genomic rearrangements, cell cycle arrest, or cell death.Recent studies have revealed that DSB in the genomes of higher eukaryotes can be repaired by at least three different pathways (2): (i) Homologous recombination repair, the most accurate process, is able to restore the original sequence at the break. Because of its strict dependence on extensive sequence homology, this mechanism is suggested to be active mainly during the S and G 2 phases of the cell cycle (3, 4). (ii) Singlestranded annealing is another homology-dependent but less accurate process that can repair DSB between direct repeats and thereby produces mainly interstitial deletions (4). (iii) Nonhomologous DNA end joining (NHEJ) comprises at least two different processes (5). The major and best investigated NHEJ pathway depends on the Ku70/80 heterodimer, the catalytic subunit of the DNA-dependent protein kinase, DNA ligase IV, and its essential co-factor XRCC4 (6, 7). In contrast to homologous recombination repair and single-stranded annealing, NHEJ can operate in the absence of sequence homology (although short sequence homologies, so-called microhomologies, may facilitate the process) and is able to rejoin broken ends directly (2). This process is supposed to occur mainly in the G 0 and G 1 phases of th...

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Section: End Joining Of 125 I-induced Double-strand Breakssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Odersky

Panyutin²,

Panyutin³

et al. 2002

Journal of Biological Chemistry

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“…The results presented here, as well as those of previous studies (4, 57, 62), indicate that the high-LET-like site-specific 125 I DSBs produced in our irradiation system are refractory to repair. The inability of HeLa WCE to efficiently end join the 125 I-induced DSBs does not appear to be due to a cofactor or enzyme deficiency of the extract, since supplementation of end joining reactions with either all four dNTPs or recombinant human DNA ligase IV/XRCC4, or both, did not improve the end joining ability of the extract (Fig.…”

Section: Discussionsupporting

confidence: 85%

“…However, based upon numerous reports (1, 8, 33, 36, 55, 56), lesions such as these might be expected to exist concurrently in combination at complex radiation-induced DSBs, and thus constitute a form of multiply damaged site. Furthermore, as we have shown here and elsewhere (4, 57), highly complex DSBs like those formed by 125 I decay, at which such multiply damaged sites do exist, are largely unrejoinable by the human NHEJ pathway. Therefore, we sought to determine if a multiply damaged DSB end consisting of a combination of lesions that are not complete blocks to NHEJ when presented individually at a DSB end, might act as a stronger block to NHEJ when presented together.…”

Section: Resultssupporting

confidence: 75%

Base Damage Immediately Upstream from Double-Strand Break Ends is a More Severe Impediment to Nonhomologous End Joining than Blocked 3′-Termini

et al. 2011

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Radiation-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions that are typically repaired by nonhomologous end joining (NHEJ) in human cells. Our previous work indicates the highly cytotoxic DSBs formed by 125I decay possess base damage clustered within 8 to 10 bases of the break, and 3′-phosphate (P) and 3′-OH ends. This study examines the effect of such structures on NHEJ in in vitro assays employing either 125I decay-induced DSB linearized plasmid DNA, or structurally defined duplex oligonucleotides. Duplex oligonucleotides that possess either a 3′-P or 3′-phosphoglycolate (PG), or a ligateable 3′-OH end with either an AP site or an 8-oxo-dG 1 nucleotide upstream (-1n) from the 3′-terminus, have been examined for reparability. Moderate to severe end-joining inhibition was observed for modified DSB ends or 8-oxo-dG upstream from a 3′-OH end. In contrast, abolition of end joining was observed with duplexes possessing an AP site upstream from a ligateable 3′-OH end, or for a lesion combination involving 3′-P plus an upstream 8-oxo-dG. In addition, base mismatches at the -1n position are also strong inhibitors of NHEJ in this system, suggesting that destabilization of the DSB terminus as a result of base loss or improper base pairing may play a role in the inhibitory effects of these structures. Furthermore, we provide data indicating that DSB end joining is likely to occur prior to removal or repair of base lesions proximal to the DSB terminus. Our results show that base damage or base loss near a DSB end may be a severe block to NHEJ, and that complex combinations of lesions presented in the context of a DSB may be more inhibitory than the individual lesions alone. In contrast, blocked DSB 3′-ends alone, are only modestly inhibitory to NHEJ. Finally, DNA ligase activity is implicated as being responsible for these effects.

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“…Given that 125 I-TFO-induced DSBs were shown to be poorly repaired in both our in vitro and in vivo DSB repair assays (Mezhevaya et al 1999;, and in light of our observation that DNA base damage proximal to a DSB end may have a significant effect upon the ability of the break to be repaired, details of the actual chemical structure of the 125 I-TFO-induced DSB would be instructive. Knowledge of the range of chemical structures associated with a 125 I-TFO-induced DSB in addition to its range of physical structure, may lead to better understanding of the biochemical requirements for its repair.…”

Section: Discussionmentioning

confidence: 93%

Characterization of a complex¹²⁵I-induced DNA double-strand break: Implications for repair

Datta

Neumann²,

Winters³

2005

International Journal of Radiation Biology

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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 > or = 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).

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Gene targeted DNA double-strand break induction by 125l-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells

Cited by 19 publications

References 59 publications

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Base Damage Immediately Upstream from Double-Strand Break Ends is a More Severe Impediment to Nonhomologous End Joining than Blocked 3′-Termini

Characterization of a complex¹²⁵I-induced DNA double-strand break: Implications for repair

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Gene targeted DNA double-strand break induction by 125l-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells

Cited by 19 publications

References 59 publications

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Repair of Sequence-specific 125I-induced Double-strand Breaks by Nonhomologous DNA End Joining in Mammalian Cell-free Extracts

Base Damage Immediately Upstream from Double-Strand Break Ends is a More Severe Impediment to Nonhomologous End Joining than Blocked 3′-Termini

Characterization of a complex125I-induced DNA double-strand break: Implications for repair

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Characterization of a complex¹²⁵I-induced DNA double-strand break: Implications for repair