ReviewDNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation
Introduction
The central role of DNA in heredity underscores the importance of genome integrity and maintenance. The enormous size of the DNA molecules comprising the chromosomes of higher eukaryotes, increases susceptibility to mechanical stress and chemical alterations. Cells counteract assaults through evolutionarily conserved mechanisms that ensure accurate DNA replication, DNA integrity and when necessary lesion removal. From the plethora of lesions induced by physical, chemical or biological agents in the DNA, double-strand breaks (DSBs) are the most deleterious and provoke cellular responses affecting almost every aspect of the cellular metabolism. They are integrated under the term DNA damage response (DDR) [1], [2], [3].
DSBs emerge in cells in a programmed manner at defined locations in the genome as part of biological processes such as meiosis, V(D)J recombination or class-switch recombination [4], [5]. They can also be generated in a random manner after exposure to agents such as ionizing radiation (IR) [6]. Depending on the inducing agent or process, DSBs can have different types of ends that influence their processing by the cell. While enzymatically induced DSBs have “clean” ends that can be directly ligated, ends generated after exposure to IR are not ligatable and require the removal of damaged nucleotides before ligation. IR induces, in addition to DSBs, also single strand breaks (SSBs) and base damages that can locally (within 10 base pairs, bp) add to a DSB, thus increasing its complexity and presumably also its reparability. This is particularly likely following exposure of cells to densely ionizing forms of radiation, such as the alpha particles emitted from the radioactive decay of radon “daughters”, or the carbon ions employed in modern radiation therapy [7], [8].
Cells of higher eukaryotes utilize two basic approaches and four pathways to process DSBs. Complete repair including restoration of molecular integrity, as well as sequence restoration in the vicinity of the DSB is achieved by homologous recombination repair (HRR). Alternatively, cells utilize an end-joining mechanism that restores integrity, but not necessarily sequence, in the DNA molecule. These mechanisms include classical non-homologous end joining (c-NHEJ), alternative end joining (alt-EJ) and, in a broader sense, single strand annealing (SSA).
In the following paragraphs we present a brief overview of the molecular components of these repair pathways, outline key processing steps and discuss pathway strengths and limitations. Subsequently, we present possible rules of pathway choice, coordination and hierarchy and discuss their implications in genomic instability—mainly by analyzing translocation formation. We conclude by describing known links between DSB repair and systemic processes manifesting as alliances between DDR and immune signaling, the regulation of specific repair pathways by growth factors and their receptors, as well as by DSB repair regulation throughout the cell cycle.
Section snippets
Homologous recombination mediated repair (HRR)
HRR is a highly evolutionarily conserved, error-free repair mechanism operating on the same principles from bacteria to human. The ends of a DSB are recognized and processed to generate 3′-single stranded DNA overhangs, through a process termed DNA end resection, or simply resection (Fig. 1).
Proteins involved in resection include Mre11, Rad50 and Nbs1, which constitute the MRN complex. Mre11 has a Mn2+ dependent single-strand endonuclease activity, as well as 3′–5′ exonuclease activity on
Single-strand annealing (SSA)
Another homology mediated DSB repair pathway, termed single-strand annealing (SSA) has been described in yeast and bacteria and may also play a role in higher eukaryotes (Fig. 3). SSA is typically initiated when DSBs occur at genomic loci where extensive homology exists between sequences at either side of the DSB and mediates the rejoining of the two ends [63]. It is conceivable, that aberrantly, it may also engage within repeat sequences in the DNA. Despite using homologies, SSA is a
Classical non-homologous end joining (c-NHEJ)
Ku is the first molecule in the cell that recognizes and binds DSBs (Fig. 4) [72], [73]—a consequence of its extremely high affinity (Kd between 2.4 × 10−9 and 5 × 10−10 M) for DNA ends [74], [75]. Ku comprises two polypeptides with sizes of about 70 and 86 kDa and has a well-characterized domain structure [74]. The resolved structure reveals a dimerization between Ku70 and Ku80 central core domains, and a ring-shaped form accommodating approximately two DNA helical turns. There are no prominent
Alternative end-joining (alt-EJ), operating as a backup (B-NHEJ)
Extensive residual repair of IR-induced DSBs in c-NHEJ deficient cells [106], but also efficient circularization of transfected viral genomes in Ku-deficient cells [107], as well as the circularization or intermolecular ligation of linearized plasmid molecules in extracts prepared from mutants lacking essential c-NHEJ activities [102], [108], suggested early on the presence of alternative end-joining mechanisms (alt-EJ) of DSB processing [109], [110]. The physiological relevance of alt-EJ is
Parameters determining DSB repair pathway choice and hierarchy
The above outline defines four main pathways implicated in DSB processing. Such repair pathway multiplicity is not known for other forms of DNA lesions. It may be evolutionarily rationalized by the severity of the DSB as a lesion and the urgency of removing DSBs from the cell genome. Collectively, the above described pathways endow cells of higher eukaryotes with an impressive potential to process DSBs. Indeed, the processing efficiency and frequently also the speed achieved tops all pathways
Cell cycle phase as determinant of DSB repair pathway activity
The above described DSB repair pathways display remarkable activity fluctuations throughout the cell cycle. Such fluctuations originate at times from fundamental repair pathway requirements that are only met in specific phases of the cell cycle. At other times, they reflect regulatory processes, which may be integrated in the circuitry of repair pathway choice.
Restriction of HRR to the S- and G2-phases of the cell cycle is commonly rationalized by the preference of this pathway for the sister
Growth-state as determinant of DSB repair pathway activity
Under standard growth conditions in vitro cultures of mammalian cells eventually exit the phase of logarithmic proliferation and enter a plateau-phase, in which cell number stagnates. This stage of reduced proliferation typically reflects limited availability, mainly through depletion, of growth factors. It is well established that when growth factors are absent from culture media, or are present at low levels, cells exit the division cycle and enter the G0-phase. This transition is reversible
Growth factor signaling in DSB repair
While the above discussed evidence implicates growth state in the efficiency of alt-EJ, direct connection with growth factor signaling is only indirectly evident. Yet, several reports suggest that mutation or overexpression of growth factor receptors modulate DDR, and there is strong evidence for interactions between growth factors receptors and components of c-NHEJ.
Thus, localized exposure to IR during cancer treatment elicits systemic effects through extracellular signals generated by
IR induced systemic effects—the immune system response
Systemic effects of IR are not limited to growth factor signaling and bystander effects. Strong effects of IR on the immune system and its cellular constituents are known and investigated for decades. IR is traditionally viewed as an immunosuppressive agent [206]. Interestingly, despite the immunosuppressive effects of high dose radiation exposure, an increasing amount of preclinical and clinical data indicate that tumor radiotherapy can, in addition to its purely cytotoxic effects, enhance
Concluding remarks
Different pathways with widely divergent strengths and limitations process DSBs in cells of higher eukaryotes. How the cell chooses among these pathways and the biochemical basis of these decisions remain to be elucidated. Yet, it is becoming increasingly evident that damage severity and constitution, location in chromatin, cell cycle phase and the proliferative state of the cell are key determining parameters. The interplay of these parameters and their relative contribution to the final
Conflict of interest
No conflict of interests.
Funding
None.
Acknowledgements
Work partly supported by DFG Graduate training program (GRK1431 and GRK1739) and grants from BMBF and BMWi.
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