Clustered DNA Double-Strand Breaks: Biological Effects and Relevance to Cancer Radiotherapy

Abstract

Cells manage to survive, thrive, and divide with high accuracy despite the constant threat of DNA damage. Cells have evolved with several systems that efficiently repair spontaneous, isolated DNA lesions with a high degree of accuracy. Ionizing radiation and a few radiomimetic chemicals can produce clustered DNA damage comprising complex arrangements of single-strand damage and DNA double-strand breaks (DSBs). There is substantial evidence that clustered DNA damage is more mutagenic and cytotoxic than isolated damage. Radiation-induced clustered DNA damage has proven difficult to study because the spectrum of induced lesions is very complex, and lesions are randomly distributed throughout the genome. Nonetheless, it is fairly well-established that radiation-induced clustered DNA damage, including non-DSB and DSB clustered lesions, are poorly repaired or fail to repair, accounting for the greater mutagenic and cytotoxic effects of clustered lesions compared to isolated lesions. High linear energy transfer (LET) charged particle radiation is more cytotoxic per unit dose than low LET radiation because high LET radiation produces more clustered DNA damage. Studies with I-SceI nuclease demonstrate that nuclease-induced DSB clusters are also cytotoxic, indicating that this cytotoxicity is independent of radiogenic lesions, including single-strand lesions and chemically "dirty" DSB ends. The poor repair of clustered DSBs at least in part reflects inhibition of canonical NHEJ by short DNA fragments. This shifts repair toward HR and perhaps alternative NHEJ, and can result in chromothripsis-mediated genome instability or cell death. These principals are important for cancer treatment by low and high LET radiation.

Keywords: DNA base damage; DNA double-strand breaks; chromatin; complex DNA lesions; cytotoxicity; genome instability; ionizing radiation; radiation oncology.

Figure 1

Relationships among DNA damage complexity, reparability, mutagenesis, and cytotoxicity. The triangles above and below indicate lesser to greater biological effects that are inversely proportional to the reparability of isolated vs. clustered DNA lesions.

Figure 2

Radiation-induced 53BP1 foci. RKO colon carcinoma cells were mock-treated or irradiated with X-rays or carbon ions. Nuclei were stained with DAPI (blue) and 53BP1 foci (red) were detected by immunofluorescence microscopy. White scale bars are 1 nm. These previously unpublished images were collected during a study comparing the induction of delayed homologous recombination by low or high LET(linear energy transfer) radiation [108].

Figure 3

Mimicking the cytotoxicity of high LET radiation with clustered DSBs(DNA double-strand breaks) induced by I-SceI nuclease. Plasmids were created with 1–4 I-SceI nuclease recognition sites spaced from 62–200 bp apart (top; N/A, not applicable). Cell lines were created with 8–14 copies of each plasmid randomly integrated into Chinese hamster ovary cells. Expression of I-SceI induces 8–48 DSBs per genome. Two cell lines were constructed with DSBs located 200 bp apart, with DSB ends either in compatible or incompatible orientation; greater killing was observed with incompatible ends (marked by *). The 4-DSB cluster showed far greater killing per DSB than low LET X-rays. The I-SceI data in this figure were adapted from a report from the Iliakis laboratory [112].

Figure 4

Key questions about clustered DSB repair, illustrated with 2-DSB clusters. (A) Short DNA fragments inhibit cNHEJ but it is unclear why distal broken ends, which are termini of long DNA fragments, are not efficiently rejoined (dashed line) by NHEJ. (B) Why do the distal ends of a DSB cluster not invade homologous sequences and repair the gap via HR? (C) If local NHEJ is delayed or fails at clustered DSBs (grey dashed lines), this may provide time for broken ends to migrate and rejoin with a broken end elsewhere in the genome by cNHEJ or aNHEJ, producing translocations. Balanced translocations are probably survivable events despite the large-scale genome rearrangement. However, unbalanced translocations create dicentric and/or acentric chromosomes that are frequently lethal. “Death*” indicates death plus other possible outcomes, such as stimulation of bridge-breakage-fusion cycles and persistent genome instability [134,135].

Figure 5

Two potential mechanisms to prevent DSB mis-repair and loss of DNA fragments at clustered DSBs. (A) Chromatin consists of DNA wrapped around nucleosomes that are highly compacted into 30 nm fibers and higher order structures. (B) Schematic of chromatin shown as a “scaffold” around DNA. (C,D) Radiation induces clustered DSBs and short DNA fragments that are prevented from mis-rejoining and/or loss by tethering factors such as DNA-PK, and by the chromatin scaffold which may act like a “splint” or “sausage skin” to prevent loss of short DNA fragments.