Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
, 11 (11)

Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

Affiliations
Review

Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

George Iliakis et al. Cancers (Basel).

Abstract

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.

Keywords: DNA repair; alt-EJ; c-NHEJ; complex DSBs; double strand breaks (DSBs); gene conversion (GC); high LET radiation; homologous recombination repair (HRR); ionizing radiation (IR); single strand annealing (SSA).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Distribution of ionizations generated in DNA-containing space by electrons of low LET, or high LET alpha particles. Note the increased ionization density from the high LET particle. When ionization events simultaneously break both DNA strands, DSBs are generated. The schematic considers for simplicity only direct IR effects, i.e., ionizations occurring directly on the DNA. However, damages generated in the DNA indirectly (indirect effects) are also very important for the overall effects of IR, i.e., by radicals produced when water molecules in the vicinity of the DNA are ionized. Such events add to the complexity of events inducing DSBs but do not fundamentally alter the rationale developed here. The contribution of indirect effects to the overall effect drops with increasing LET of the radiation used.
Figure 2
Figure 2
(a) Repair of DSBs by DNA end resection independent and DNA end resection dependent DSB repair pathways. Classical non-homologous end joining (c-NHEJ) is initiated by the strong binding of KU heterodimer and the recruitment of DNA-PKcs protein kinase, which is believed to block and attenuate DNA end resection dependent DSB processing. Gene conversion (GC), single-strand annealing (SSA), and alternative end joining (alt-EJ) are initiated by precisely controlled DNA end resection. Only GC results in faithful restoration of the DNA sequence around the DSB. All remaining DSB repair pathways are error-prone and are characterized by the generation of genomic alterations. The possible ramifications of the distinct characteristics of the different DSB repair pathways and the significance of their error-prone nature are discussed in the text. (b) Outline of the cell cycle dependence of the above DSB processing pathways.
Figure 3
Figure 3
(a) Representative survival experiments with DNA-PKcs-proficient (M059K) and DNA-PKcs-deficient (M059J) cells exposed to low LET (x-rays) and high LET (56Fe+ ions, LET = 151 keV/µm). Note that c-NHEJ deficiency associated with the DNA-PKcs defect eliminates the LET-dependent radiosensitization observed with wild-type cells. The results depicted here are re-drawn from Singh et al. to illustrate the concepts developed in the present review. (b) Similar results obtained with the XRCC2-deficient irs1 mutant of V79 that generates a strong defect in GC. The results shown have been traced from Hill et al.
Figure 4
Figure 4
Pulsed-field gel electrophoresis (PFGE) experiments of c-NHEJ-proficient (M059K) and c-NHEJ-deficient (M059J) cells exposed to x-rays and 56Fe+ ions (LET 151 keV/µm). (a) Dose response curves of M059K and M059J cells exposed to low or high LET IR. Note that the induction of DSBs is not affected by the LET or the defect in c-NHEJ. (b) Repair kinetics of M059K and M059J cells exposed to low or high LET IR. Note that despite subtle differences, c-NHEJ-proficient and -deficient cells actively repair DSBs after exposure to either high or low LET IR.
Figure 5
Figure 5
Concepts utilized in the classification of DSBs according to their degree of complexity. REs-generated DSBs break the DNA without chemically altering its nucleotides and are therefore considered the simplest form of DSBs (upper left box). IR induces DSBs by chemically altering at minimum two nucleotides in opposite strands mainly from ionization events within an ionization cluster (lower left box). The chemical entities generated in this way at the ends vary and need to be removed during DSB processing. This DSB is therefore more “complex” than that generated by REs. Since an ionization cluster may contain more than two events, the DSBs generated may be accompanied by additional forms of DNA damages near the DSB in the form of either additional single strand breaks or base damages. This generates DSBs of higher “complexity” in the sense that they will need more extensive processing (lower right box). Occasionally, particularly when cells are exposed to high LET particles, multiple DSBs are generated in some proximity (upper right box). The chromatin fragments generated in this way may be lost and are likely therefore to destabilize chromatin and compromise DSB processing. We therefore define them here as an additional level of DSB complexity. The consequences of defined generation of DSB clusters in cells are discussed in detail in the text.
Figure 6
Figure 6
A model system to study the biological consequences of defined DSB clusters in Chinese Hamster Ovary (CHO) cells using engineered clusters of I-SceI recognition sequences. (a) Schematic representation of I-SceI constructs containing single DSBs, DSB pairs, and DSB quadruplets and integrated multiple times in the genome of CHO cells. (b) Description of the cell lines generated explaining the nomenclature used. The number of I-SceI sites (1×, 2×, 4×), their orientation (direct-D, reverse-R), and the number of integrations is presented in the table. (c) Clonogenic survival experiments of CHO cells harboring DSB clusters of different complexity. (d) Translocations forming in the same cell lines when analyzed at metaphase without any treatment, or after treatment with the PARP-1 inhibitor, PJ34. (e) Relative increase in chromosomal translocations after inhibition of c-NHEJ by incubating with the DNA-PKcs inhibitor NU7441. The results shown have been published and are redrawn here to illustrate the concepts developed in the review [57].

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Kuppers R. Mechanisms of B-cell lymphoma pathogenesis. Nat. Rev. Cancer. 2005;5:251–262. doi: 10.1038/nrc1589. - DOI - PubMed
    1. Lieber M.R. Mechanisms of human lymphoid chromosomal translocations. Nat. Rev. Cancer. 2016;16:387–398. doi: 10.1038/nrc.2016.40. - DOI - PMC - PubMed
    1. Bunting S.F., Nussenzweig A. End-joining, translocations and cancer. Nat. Rev. Cancer. 2013;13:443–454. doi: 10.1038/nrc3537. - DOI - PMC - PubMed
    1. Schipler A., Iliakis G. DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice. Nucleic Acids Res. 2013;41:7589–7605. doi: 10.1093/nar/gkt556. - DOI - PMC - PubMed
    1. Mladenova V., Mladenov E., Iliakis G. Novel Biological Approaches for Testing the Contributions of Single DSBs and DSB Clusters to the Biological Effects of High LET Radiation. Front. Oncol. 2016;6:163. doi: 10.3389/fonc.2016.00163. - DOI - PMC - PubMed
Feedback