There are many external agents that can lead to DSBs in a cell. The most well known are high-energy radiation in the form of gamma rays, alpha particles or beta rays. All these high energy radiation sources will either directly shatter DNA or lead to the formation of an ionization trail with highly reactive molecules such as ROS, which will in turn attack and react with DNA causing DSBs. The most effective radiation in producing DSBs are alpha particles, which deposit a high amount of energy in the form of ionization events over a very short distance in the cell.

In addition to leading directly to a DSB as a consequence of the action of ROS or a direct shattering of the DNA strands, radiation will also generate interstrand crosslinks which will cause a problem during replication (see below).

ROS are normally produced in a cell as the result of metabolic processes (Sallmyr et al. 2008). If ROS are not scavenged efficiently or produced at a higher rate due to increased metabolic activity, the likelihood that DSB are generated increases.

DNA replication can be a major source of DSB breaks. The replication fork stalls if it encounters a mismatch or an intrastrand crosslink. Attempts to repair mismatches generated during replication can lead to abortive repair by the mismatch repair pathway which will lead to DSBs. As mentioned above, the frequency of mismatches is increased if the nucleotide pools are depleted due to rapid proliferation causing the DNA polymerases to incorrectly incorporate nucleotides at a higher rate (Bester et al. 2011).

Interestingly, there are several physiological processes which introduce DSBs “on purpose”. These processes are required for the generation of genomic rearrangements at the immunoglobulin and the T cell receptor loci during the maturation of B and T cells, respectively (Dudley et al. 2005). The mechanisms that lead to these DSBs and their proper resolution in the form of productive rearrangements have been studied in great detail and their involvement in the generation of chromosomal translocations that drive lymphoid malignancies has been subject to intense scrutiny (Zhang et al. 2010).

The RAG1/2 recombination-associated endonucleases are responsible for introducing DSB to initiate the process of VDJ recombination at IGH, TRB, and TRD loci and the VJ recombination process at the IGK, IGL, TRA and TRG loci. The DSBs introduced by the RAGs are the first step in recombining one of many V (variable) segments with one of many J (joining) segments to create functional immunoglobulin or T cell receptor genes. In the case of the IGH, TRB and TRD loci a D (diversity) segment to J segment recombination precedes the V–J recombination for a full VDJ recombination event. The V(D)J recombination is completed by repairing or joining the DSBs with the classical nonhomologous end-joining pathway (C-NHEJ) (Zhang et al. 2010) (see below).

DSB are very dangerous for a cell because they compromise the integrity of its DNA. Since the purpose of these DSBs is to produce a functional gene, the RAG endonucleases do not cut randomly in the genome but are guided by specific recombination signal sequences (RSS), which consist of heptamer/nonamers that are separated either by a 12 bp or a 23 bp spacer (Tonegawa 1983). The RAGs only introduce DSB in two heptamer/nonamer sequences if one contains a 12 bp and the other a 23 bp spacer. Even though there is sequence specificity and several constraints in where DSBs can be introduced by the RAGs, some variations in the heptamer/nonamer sequences are tolerated. This means that DSBs can be introduced at other genomic loci that happen to have sequences which resemble the heptamer/nonamer sequences of the immunoglobulin and TCR loci. It is quite obvious that DSBs outside the immunoglobulin or TCR loci can lead to unintended and potentially dangerous chromosomal rearrangements if joined via the C-NEHJ pathway (Robbiani et al. 2009).

In addition to the diversity in the immunoglobulin and TCR genes generated by the V(D)J recombination process, more diversity is achieved by the process of somatic hypermutation (SHM). In SHM, mutations are introduced into the variable region exons of the immunoglobulin or TCR genes. The key enzyme responsible for SHM is the activation-induced cytidine deaminase (AID). AID deaminates cytidines in single-stranded DNA regions. The AID enzyme does not only introduce mutations into the variable region exons (Li et al. 2004) but is also responsible for the DSB required for class switch recombination (CSR) (Muramatsu et al. 2000). Since AID needs single-stranded DNA, its activity is coupled to active transcription which requires the unwinding and melting of the DNA double strand. Thus, AID is targeted by active transcription to the switch region of the constant region exons (for CSR) or to the variable region exons (for SHM). The cytidine deamination events introduced by AID into the switch regions of the constant region exons are processed to DSB through the activity of proteins involved in DNA repair (Chaudhuri and Alt 2004; Di Noia and Neuberger 2007). What causes AID induced deamination events to lead to single nucleotide mutations and small indels at the variable region exons on the one hand and to DSB in the switch regions of the constant region exons on the other hand is currently unknown (Muramatsu et al. 2007). High throughput genome-wide translocation sequencing showed that AID-associated DSBs that occur outside the normal loci for CSR are more frequent in actively transcribed regions (Chiarle et al. 2011).

The nucleus is a very crowded place occupied by 46 linear DNA molecules with a total length of about 2 m crammed into a small space of just a few cubic micrometers (approximately 100–200 µm³). Many processes in the nucleus like transcription, DNA replication and chromosome condensation require the unwinding of supercoiled DNA to relieve torsional stress and to allow replication and transcription factors to gain access to specific sites. Due to topological constraints it frequently becomes necessary to break one DNA double strand temporarily to pass another DNA strand through this gap. Introducing a DSB into a DNA molecule to pass another DNA strand through the gap and then repairing the gap is a very risky process and can easily lead to a faulty repair if another DSB is in the vicinity. Topoisomerases and helicases and a number of DNA repair proteins are involved in this process (Kaneko et al. 2004; Plank and Hsieh 2009; Vos et al. 2011).

To relieve torsional stress of a DNA molecule, e.g., during transcription, DNA helicases will introduce a nick in one strand of a dsDNA molecule, release the stress and then re-ligate the single-stranded nick. This process is also risky since it can result in a DSB (Carrasco et al. 2014).

The first detectable event after a DSB is poly(ADP-ribosyl)ation (PAR) mediated by the PARP1-3 enzymes. PAR modifies lysine residues of the core histones tails. PAR of the histone tails leads to the recruitment of the chromatin remodelling complex NuRD/CHD4 and polycomb complexes (Chou et al. 2010).

Another early event at a DSB is the phosphorylation of histone H2AX by ATM to form gamma-H2AX. Gamma-H2AX is recognized by its sensor MDC1 (mediator of DNA damage checkpoint protein1), which interacts among others, with the Nijmegen breakage syndrome protein, NBS1 (Goldberg et al. 2003). NBS1 in turn interacts with ATM and tethers it to the DSB to increase local gamma-H2AX concentration. MDC1 recruits a number of other proteins like RNF8 which subsequently leads to the recruitment of BCRA1, RAD18, PTIP (Pax transactivation domain interacting protein) and P53BP1 to the sites of DSBs (Lukas et al. 2011).

It should be noted that replication stress in the form of stalled replication forks also elicit gamma-H2AX formation via ATR (ATM and Rad 3-related) kinase activity (Wang et al. 2011).

The TP53 tumor suppressor gene plays an important role in the detection and signaling of DNA damage in a cell and orchestrating cellular responses to DNA damage like cell cycle control and apoptosis TP53 (Meek 2009).

P53BP1 (P53 binding protein 1) can shield under-replicated DNA regions after the cell has gone through mitosis from the activity of DNA nucleases until the under-replicated DNA regions have been repaired. These under-replicated DNA regions, which are often found at common fragile sites, can become visible in mitosis as so-called ultrafine DNA bridges. 53BP1 nuclear bodies are found in the nuclei of cells that have just passed through mitosis (Lukas et al. 2011).

It should be noted that the exact molecular mechanisms that lead to DSB recognition are far from being completely understood and the description above offers only a glimpse of the complexity that is already known. These mechanisms also vary from cell type to cell type and also depend on the origin of the DSBs.

Homologous recombination is a usually error free DSB repair pathway. This process operates in the S and G2 phase of the cell cycle because it requires an intact sister chromatid, which serves as the template for the DNA repair. The MRE11/RAD50/NBS protein complex is loaded onto the end of the DSBs where the 5′-3′ exonuclease activity of MRE11 removes nucleotides from one of the strands of the free dsDNA end (Daley et al. 2013; Lammens et al. 2011; Williams et al. 2010). This leads to an exposed single-stranded 3′ DNA overhang, which is processed with the help of replication proteins A (RPA), RAD51, BRCA2 and several other proteins into the RAD51-ssDNA-nucleoprotein filament. The RAD51-ssDNA-nucleoprotein filament will then invade the dsDNA of the homologous site on the sister chromatid, which serves as a template for the error-free synthesis of the DNA across the DSB region (Pellegrini et al. 2002; Popp and Bohlander 2010; Yang et al. 2002). After the Holliday junctions are resolved, the error-free repair is completed. This repair pathway can result in sister chromatid exchange. The DSB repair by HR is a sterically very complicated process and involves, in addition to the proteins mentioned above, several other important proteins like RECQL2 (Werner Syndrome), BLM (RECQL3) (Bloom Syndrome), BRCA2 (familial breast cancer), RAD54, PALB2 (familial pancreatic cancer), FANCM, FANCC (Fanconi anemia) and others (Daley et al. 2013; Popp and Bohlander 2010). A number of these proteins are mutated in hereditary tumor or genome instability syndromes and are also somatically mutated in these tumors (Bunting and Nussenzweig 2013; Ellis et al. 1995; Jones et al. 2009; Meetei et al. 2003; Strathdee et al. 1992; Wooster et al. 1995; Yu et al. 1996).

SSA is a variant of HR, a homology dependent mode of joining DSB. It operates between closely spaced direct repeats (less than 25 kbp apart) and usually results in the deletion of one copy of the repeat (Ivanov et al. 1996; Zhang et al. 2010).

Another, mechanism to generate a translocation would be for the DNA replication machinery to switch to another chromosome as a template. This so-called breakage-induced replication (BIR) template switching can be induced by a break during DNA replication (Bunting and Nussenzweig 2013).

The C-NHEJ repair pathway for DSB is error-prone, which means that after the joining of the broken ends there will be, mostly smaller, deletions or insertions at the location of the former DSB (Rassool 2003; Roth and Wilson 1986). This process does not need an intact template strand from the sister chromatid and is therefore the major repair pathway for DSBs during the G1 and S phases of the cell cycle. The C-NHEJ pathway is also far more likely to join DSBs that do not belong together since it does not require homologous sequences to guide the repair process. It is, together with alternative end joining (A-EJ), the major repair pathway for removing DSB in the cells. C-NHEJ is used in V(D)J recombination at the immunoglobulin and T cell receptor loci for repairing the breaks that are generated in the process of somatic recombination and CSR.

To initiate the C-NHEJ process, the DSB DNA ends are bound by the KU70/80 proteins to prevent the drifting apart of the DSB ends (Soutoglou et al. 2007). Then DNA-PKCs and the MRN complex (MRE11-RAD50-NBS) are recruited to the breaks. The MRN complex is crucial in all three DSB repair processes (HR, C-NHEJ and A-EJ). It executes and coordinates many of the activities that are required for DSB repair. For example, the RAD50 coiled-coil domains extend from the core MRN complex which occupies the DSB end to connect the DSB to the other break or the sister chromatid (Lammens et al. 2011).

The MRN complex is responsible for some resection at the DSB and then the broken DNA ends are joined together by the activity of the DNA ligase IV-XRCC4 complex (Roth and Wilson 1986).

Other important proteins involved in C-NHEJ are Artemis (Moshous et al. 2001) and Cerunnos (XLF) (Ahnesorg et al. 2006; Buck et al. 2006). Both proteins were identified because mutations in these factors lead to certain severe combined immunodeficiency syndromes, in which immunoglobulin recombination and TCR recombination are severely compromised.

There are probably several A-EJ pathways. A-EJ is the repair of DSB in the absence of KU70/80, XRCC4 or Ligase 4 (Daley and Wilson 2005; Wang et al. 2003). The A-EJ pathway also relies on the MRN complex as the crucial component of the repair process (Popp and Bohlander 2010).

A-EJ repaired DSBs are characterized by the presence of stretches of microhomologies of six to eight base pairs. In some models, A-EJ pathways are responsible for the majority of DSB repairs that resulted in chromosomal translocations (Boboila et al. 2010; Simsek and Jasin 2010).

In systems with inducible chromosomal translocations, the analysis of the breakpoints showed that microhomology-based mechanisms were responsible for only a minority of de novo translocations (Daley and Wilson 2005; Wang et al. 2003). Next generation sequencing studies also showed that less than a third of human germ line chromosomal translocations showed microhomologies at the breakpoints. The breakpoints showed more complicated structures with fragmentation of local DNA sequences, small inversions and deletions. This suggests that C-NHEJ rather than A-EJ contributes to a substantial fraction of human germline chromosomal translocations (Chiang et al. 2012).