**2. Effects of ionizing radiation on cells and cell nuclei**

Ionizing radiation penetrates through material and deposits enough energy to ionize molecules or atoms by liberating electrons. The effects of ionizing radiation on biological materials are highly dependent on the dose, the dose rate and type of radiation. In living cells, ionizing radiation hits all kinds of biomolecules, such as desoxyribonuleic acids (DNAs), aminoacids (proteins), lipids (membranes), carbohydrates, etc. However, most harmful consequences to living organisms show damages inflicted to their genomic DNA, especially in the form of DNA double-strand breaks (DSBs) [25, 26]. Especially follow-up effects of false strand repair may lead to significant dysfunctional development as for instance tumor genesis.

**45**

*Super-Resolution Radiation Biology: From Bio-Dosimetry towards Nano-Studies of DNA Repair…*

Ionizing radiation (IR) includes all high energy/speed (> 1% speed of light) ions (e.g. carbon ions), atom nuclei (e.g. alpha particles), subatomic particles (e.g. beta particles, protons or neutrons) and high-energy electromagnetic waves (e.g. high energy ultraviolet (UV) rays, X-rays and gamma rays), that carry enough energy to directly or indirectly ionize atoms or molecules by liberating electrons from them,

The most common types of ionizing radiation occurring under environmental circumstances are caused by radioactive decay and can be divided into three groups: alpha, beta and gamma radiation [27]. Alpha radiation is made up of particles comprising two protons and two neutrons (helium nucleus) that carry energies in the range of up to several MeV. Due to its large particle size, alpha radiation has the lowest penetration depth through biological materials and the highest energy deposition per distance traveled. Beta particles are made up of electrons or positrons, thus exerting higher penetration depths and lower energy depositions compared to alpha particles. Gamma rays are high energy electromagnetic waves that exhibit the highest penetration and compared to particles, lowest energy deposition per track unit in biomaterials among these three types of IR. Due to the dispersed and low energy deposition in tissue, gamma and beta radiation are often referred to as low linear energy transfer (LET) radiation, whereas alpha particles belong to high LET

For clinical diagnosis and therapy in radiology or radiation oncology [28, 29], typically artificial radiation sources are applied, as for instance to produce X-rays in the energy range of keV to MeV, electrons and positrons, protons, and heavy ions (carbon or nitrogen). Like alpha particle, protons and heavy ions belong to high LET radiation. The advantage of protons and especially heavy ions is based on the characteristic absorbance with a Bragg peak at the end of the particle track where most of the particle energy is deposited. This energy positioning peak can exactly be localized in the tumor volume so that intact cells and tissues in the tumor

The absorbed dose D of ionizing radiation is quantified by the amount of energy deposited per unit mass of the penetrated material and is measured in units of Joule per kilogram (J/kg) or Gray (Gy) [27]. It describes an universal energy absorption for all types of ionizing radiation and is most commonly used in radiophysical research, whereas a radiation type specific dose also called the equivalent dose H calculated by multiplication with a weighting factor WR (e.g. WR = 1 for gamma radiation and WR = 20 for alpha radiation) is often used in radio-biology, radio-medicine, or radiation protection and safety. The equivalent dose can be further weighted by a tissue weighting factor WT to result in the effective dose E, which describes radiobiological effects considering the used radiation type and the tissue/organ of interest. Both, the equivalent dose and effective dose are quantified in units of Sievert (Sv) and do not represent physically measurable quantities but rather a value based on clinical and epidemiological outcome that is typically used

Among all kinds of ionizing radiation induced biological effects, damages to chromatin especially the DNA molecules in the nucleus of cells are thought to be the

surroundings are excluded from radiation damaging [30].

**2.3 Damages of DNA induced by ionizing radiation**

*DOI: http://dx.doi.org/10.5772/intechopen.95597*

and to break molecular bonds [27].

**2.1 Ionizing radiation**

radiation [27].

**2.2 Dose measures**

in radiation safety [31].

*Super-Resolution Radiation Biology: From Bio-Dosimetry towards Nano-Studies of DNA Repair… DOI: http://dx.doi.org/10.5772/intechopen.95597*

### **2.1 Ionizing radiation**

*DNA - Damages and Repair Mechanisms*

oncology, radiation therapy planning etc.) research strongly contributed to the current understanding of ionizing radiation effects on human organs, tissues, and cells [1, 2]. In principle radiation biology is based on effects of instantaneous (10−18 s) [3], stochastic damaging interactions of ionizing radiation with cells, a main target being the genetic material, i.e. chromatin in the cell nucleus [4]. In this context, radio-sensitivity and radio-resistance as opposing terms describe the extent of individual cellular susceptibility or 'response' upon radiation exposure which are highly dependent on physical (e.g., radiation type, dose, dose rate, etc.), chemical (e.g., hydroxyl radicals, etc.) and biological (e.g., developmental and proliferative state of the affected cell type) factors. As the overall organismal radiation response results from the entirety of all individual radiation responses on the single cell level, deeper understanding of the underlying, complex molecular mechanisms and dynamics of radiation induced DNA damaging and repair on the cellular level is highly relevant

for fundamental and applied radiation biology (for review see [1, 2, 5]).

Hence, cytometric analyses based on fluorescence microscopy have become the method of choice to study damaging effects of ionizing radiation and DNA repair. This has contributed a lot to today's knowledge. However, conventional fluorescence microscopy is limited to average lateral resolutions around 200 nm laterally and 600 nm axially [6] and thus is limited to the bulk analysis of molecular cellular processes and structures. In parallel, super-resolution fluorescence microscopy techniques have rapidly evolved during the last few decades and turned out to be powerful tools to study cellular structures and molecular architectures on the nanoscale (for review see [6–8]). Methods based on stochastic reversible photobleaching [9–15] of single molecules called Single Molecule Localization Microscopy (SMLM) [16] reach effective resolutions down to 10 nm and have become popular among modern super-resolution imaging techniques as their realization is highly practical and straightforward using established specimen preparation methods of standard fluorescence microcopy [17]. As such resolutions allow the detection of single molecules, such as nucleosomes [18], proteins [19, 20], receptors and junction proteins [21, 22], or even single chromatin loops [23] etc., super-resolution microscopy opens new avenues for the research of radiation induced damaging and repair

With this article, we attempt to introduce the novel SMLM approach to radiation biophysics and radiation biology. We start with a brief summary on the basics of ionizing radiation, induction of DNA damage and damage repair mechanisms, to follow up with some standard radiobiology analysis methods. We further provide an overview of the working principles of selected sub-diffraction microscopy techniques with a focus on SMLM. Finally, the successful application of localization microscopy in radiation biology research is demonstrated along examples of

Ionizing radiation penetrates through material and deposits enough energy to ionize molecules or atoms by liberating electrons. The effects of ionizing radiation on biological materials are highly dependent on the dose, the dose rate and type of radiation. In living cells, ionizing radiation hits all kinds of biomolecules, such as desoxyribonuleic acids (DNAs), aminoacids (proteins), lipids (membranes), carbohydrates, etc. However, most harmful consequences to living organisms show damages inflicted to their genomic DNA, especially in the form of DNA double-strand breaks (DSBs) [25, 26]. Especially follow-up effects of false strand repair may lead

**2. Effects of ionizing radiation on cells and cell nuclei**

to significant dysfunctional development as for instance tumor genesis.

**44**

processes [5, 24].

current works.

Ionizing radiation (IR) includes all high energy/speed (> 1% speed of light) ions (e.g. carbon ions), atom nuclei (e.g. alpha particles), subatomic particles (e.g. beta particles, protons or neutrons) and high-energy electromagnetic waves (e.g. high energy ultraviolet (UV) rays, X-rays and gamma rays), that carry enough energy to directly or indirectly ionize atoms or molecules by liberating electrons from them, and to break molecular bonds [27].

The most common types of ionizing radiation occurring under environmental circumstances are caused by radioactive decay and can be divided into three groups: alpha, beta and gamma radiation [27]. Alpha radiation is made up of particles comprising two protons and two neutrons (helium nucleus) that carry energies in the range of up to several MeV. Due to its large particle size, alpha radiation has the lowest penetration depth through biological materials and the highest energy deposition per distance traveled. Beta particles are made up of electrons or positrons, thus exerting higher penetration depths and lower energy depositions compared to alpha particles. Gamma rays are high energy electromagnetic waves that exhibit the highest penetration and compared to particles, lowest energy deposition per track unit in biomaterials among these three types of IR. Due to the dispersed and low energy deposition in tissue, gamma and beta radiation are often referred to as low linear energy transfer (LET) radiation, whereas alpha particles belong to high LET radiation [27].

For clinical diagnosis and therapy in radiology or radiation oncology [28, 29], typically artificial radiation sources are applied, as for instance to produce X-rays in the energy range of keV to MeV, electrons and positrons, protons, and heavy ions (carbon or nitrogen). Like alpha particle, protons and heavy ions belong to high LET radiation. The advantage of protons and especially heavy ions is based on the characteristic absorbance with a Bragg peak at the end of the particle track where most of the particle energy is deposited. This energy positioning peak can exactly be localized in the tumor volume so that intact cells and tissues in the tumor surroundings are excluded from radiation damaging [30].

#### **2.2 Dose measures**

The absorbed dose D of ionizing radiation is quantified by the amount of energy deposited per unit mass of the penetrated material and is measured in units of Joule per kilogram (J/kg) or Gray (Gy) [27]. It describes an universal energy absorption for all types of ionizing radiation and is most commonly used in radiophysical research, whereas a radiation type specific dose also called the equivalent dose H calculated by multiplication with a weighting factor WR (e.g. WR = 1 for gamma radiation and WR = 20 for alpha radiation) is often used in radio-biology, radio-medicine, or radiation protection and safety. The equivalent dose can be further weighted by a tissue weighting factor WT to result in the effective dose E, which describes radiobiological effects considering the used radiation type and the tissue/organ of interest. Both, the equivalent dose and effective dose are quantified in units of Sievert (Sv) and do not represent physically measurable quantities but rather a value based on clinical and epidemiological outcome that is typically used in radiation safety [31].

#### **2.3 Damages of DNA induced by ionizing radiation**

Among all kinds of ionizing radiation induced biological effects, damages to chromatin especially the DNA molecules in the nucleus of cells are thought to be the most severe with respect to cellular survival and carcinogenesis [2, 5, 32, 33]. DNA base oxidation, single strand breaks (SSBs) and double strand breaks (DSBs) are the most common ionizing radiation induced damages to the DNA molecule, that affect genome integrity and DNA biochemistry [34].

DSBs of DNA belong to the most complex and severe types of DNA damages as they directly affect genome integrity and the way of cellular survival [35–37]. Single strand breaks (SSBs) induced by ionizing radiation and base damages occur more frequently than double strand breaks [34]. It can be estimated to about 40 DSBs/Gy and about 1,000 SSBs/Gy. SSBs are less severe to genome integrity as an intact template strand is still available for complementarity-aided, error-free repair of the lesion. But DSBs are also simply formed by two or more opposing SSBs in close proximity or combinations of different DNA damage types [26].

Induction of DSBs in native chromatin is rapidly followed up by phosphorylation of nearby histones of the H2A variant H2AX at serine residues at position 139 [38]. This results in the generation of plenty γH2AX molecules around a DSB damage site, where about 2 Mbp of DNA are usually phosphorylated [39]. This leads to the formation of focus structures of sizes in the range of micrometers, which can be visualized under a fluorescence microscope [40]. These phosphorylated histones serve as signal and anchor points for many downstream recruited proteins of certain DNA damage response and repair machineries [41]. As the number of γH2AX foci is quantitative for DNA damage, counting of specifically labeled foci has been established as a measure for dose-efficiency and correlated to cell survival [42].

Single ionizing radiation induced DNA lesions can be caused by direct or indirect hits [43]. Ionizing radiation penetrating through a cell nucleus can hit and ionize atoms in a DNA molecule itself with a certain probability. However, the most prominent primary reaction underlying all ionizing radiation induced DNA damages is the radiolysis mediated formation of reactive oxygen species (ROS), e.g. •OH radicals, O2•- radicals and H2O2, which can further inflict reducing damage and thus lesion to the DNA [44]. Ionizing radiation, especially high LET radiation, is known for its property to efficiently induce highly complex damages to DNA. Such complex DNA damage sites composed of multiple lesions in close proximity on both strands are also termed locally multiple damage sites (LMDS) [45].

## **3. DNA double strand break repair mechanisms**

Living organisms developed highly efficient and customized ways to repair the severe damages inflicted to their genome. The DNA DSB sites are rapidly (within seconds to minutes) recognized and marked by proteins of an initial response, which serve as signals and docking sites for more specialized proteins of DNA repair pathways. The fate of repair type depends on the concerted presence of pathway specific damage response proteins [1, 2, 46–48]. The main two ways by which cells respond to DNA double-strand breaks are non-homologous end joining (NHEJ; also called canonical NHEJ = cNHEJ) and homologous recombination (HR). NHEJ mediated DSB repair is fast and can be error-prone, but it can be flexibly performed throughout all cell cycle phases. HR works error-free, but is mostly restricted to late S and G2 phases as a homologous sister chromatid is required as a repair template [49–52]. Recent data, however, have suggested that active genes may employ HR also in G1 phase, by utilizing the nascent RNA as a template for precise repair (reviewed in [53]). As the DNA-end resection is inhibited in G1 cells, an alternative model with cNHEJ taking the advantage of the same principle (RNA-templated repair) has also been proposed. Interestingly, DNA repair by HR is preferred in lower eukaryotic life forms, whereas NHEJ is predominantly observed

**47**

*Super-Resolution Radiation Biology: From Bio-Dosimetry towards Nano-Studies of DNA Repair…*

in mammalian organisms. Alternative low abundant DSB repair pathways are the alternative end joining pathways (a-NHEJ; also called back-up EJ), micro-homology mediated EJ (MMEJ) and single strand annealing (SSA). One main difference between all DSB repair mechanisms is the extent of initial DNA end-resection at the damage site [26, 54–59]. The DNA damage response (DDR) against DSBs is subject to intensive radiobiological investigation and fluorescence microscopy of in situ

After the induction of a DSB, damage response proteins are rapidly recruited and accurately determine the fate of the DSB towards a repair pathway that best deals with the damage site in a certain genomic and cellular context. The chromatin remodeling p53-binding protein (53BP1) protects the break site from extensive end resection [60], thereby promoting repair by non-homologous end joining [61], whereas BRCA1 facilitates extensive end resection for repair by homologous

The NHEJ repair pathway is initiated with binding of the Ku70-Ku80 heterodimer complex to the DNA ends of the DSB site, which serves as a linkage between damage site and further damage response proteins [61, 63]. In a second step, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to the Ku complex forming the DNA-PK complex. On-going recruitment of X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF to the DNA-PK complex forms the core NHEJ complex [64]. DNA-PKcs sterically protects the break site for repair and phosphorylates other repair proteins [65, 66] and H2AX [41]. Furthermore, DNA-PK auto-phosphorylation results in a conformational change of the core complex, thereby enabling DNA end processing by nucleases and dissociation of the DNA-PKcs subunit [67, 68]. Finally, ligation of the DNA ends is mediated by the X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF [69–71]. Artemis endonuclease [72, 73], polynucleotide kinase (PNK) [74], DNA polymerase (pol) μ and λ can be additionally involved in NHEJ repair depending on

To initiate repair by HR, the free damaged DNA ends at the DSB site must be sensed and bound by a protein complex comprised of MRE11, RAD50 and NBS1 (MRN complex) [76]. Next, the protein kinase Ataxia Telangiectasia Mutated (ATM) [77] is recruited to the MRN complex at the damage site [78], which autophosphorylates and phosphorylates components of the neighboring chromatin. Most prominent phosphorylations are those of the histone variant H2AX (γH2AX), one of the earliest and a very sensitive marker of cellular response to DSBs [38]. End resection is initiated by the single-strand endonuclease and exonuclease activity of the Mre11 protein [52, 79] of the MRN complex. RAD50 further stimulates Mre11 nuclease activity and Nbs1 interacts with CtIP [80], another protein that is essential for the initiation of MRN complex mediated end resection [81]. Exonuclease 1 (Exo1) and Dna1/BLM are recruited by CtIP to continue end resection [82–84] until it gets attenuated by RPA coating of resected ssDNA ends [85]. BRCA2 in combination with BRCA1 and PALB2 dismantles the ssDNA ends from RPA coats enabling binding and forming of the RAD51 nucleoprotein filament, which stimulates

DSB repair proteins serves as state of the art biological dosimetry.

*DOI: http://dx.doi.org/10.5772/intechopen.95597*

**3.1 The initial response**

recombination [52, 62].

**3.2 Non-homologous end joining**

**3.3 Homologous recombination**

the chemical properties of the DNA damage site [75].

*Super-Resolution Radiation Biology: From Bio-Dosimetry towards Nano-Studies of DNA Repair… DOI: http://dx.doi.org/10.5772/intechopen.95597*

in mammalian organisms. Alternative low abundant DSB repair pathways are the alternative end joining pathways (a-NHEJ; also called back-up EJ), micro-homology mediated EJ (MMEJ) and single strand annealing (SSA). One main difference between all DSB repair mechanisms is the extent of initial DNA end-resection at the damage site [26, 54–59]. The DNA damage response (DDR) against DSBs is subject to intensive radiobiological investigation and fluorescence microscopy of in situ DSB repair proteins serves as state of the art biological dosimetry.

### **3.1 The initial response**

*DNA - Damages and Repair Mechanisms*

affect genome integrity and DNA biochemistry [34].

most severe with respect to cellular survival and carcinogenesis [2, 5, 32, 33]. DNA base oxidation, single strand breaks (SSBs) and double strand breaks (DSBs) are the most common ionizing radiation induced damages to the DNA molecule, that

DSBs of DNA belong to the most complex and severe types of DNA damages as they directly affect genome integrity and the way of cellular survival [35–37]. Single strand breaks (SSBs) induced by ionizing radiation and base damages occur more frequently than double strand breaks [34]. It can be estimated to about 40 DSBs/Gy and about 1,000 SSBs/Gy. SSBs are less severe to genome integrity as an intact template strand is still available for complementarity-aided, error-free repair of the lesion. But DSBs are also simply formed by two or more opposing SSBs in

Induction of DSBs in native chromatin is rapidly followed up by phosphorylation of nearby histones of the H2A variant H2AX at serine residues at position 139 [38]. This results in the generation of plenty γH2AX molecules around a DSB damage site, where about 2 Mbp of DNA are usually phosphorylated [39]. This leads to the formation of focus structures of sizes in the range of micrometers, which can be visualized under a fluorescence microscope [40]. These phosphorylated histones serve as signal and anchor points for many downstream recruited proteins of certain DNA damage response and repair machineries [41]. As the number of γH2AX foci is quantitative for DNA damage, counting of specifically labeled foci has been established as a measure for dose-efficiency and correlated to cell survival [42]. Single ionizing radiation induced DNA lesions can be caused by direct or indirect hits [43]. Ionizing radiation penetrating through a cell nucleus can hit and ionize atoms in a DNA molecule itself with a certain probability. However, the most prominent primary reaction underlying all ionizing radiation induced DNA damages is the radiolysis mediated formation of reactive oxygen species (ROS), e.g. •OH radicals, O2•- radicals and H2O2, which can further inflict reducing damage and thus lesion to the DNA [44]. Ionizing radiation, especially high LET radiation, is known for its property to efficiently induce highly complex damages to DNA. Such complex DNA damage sites composed of multiple lesions in close proximity on both

close proximity or combinations of different DNA damage types [26].

strands are also termed locally multiple damage sites (LMDS) [45].

Living organisms developed highly efficient and customized ways to repair the severe damages inflicted to their genome. The DNA DSB sites are rapidly (within seconds to minutes) recognized and marked by proteins of an initial response, which serve as signals and docking sites for more specialized proteins of DNA repair pathways. The fate of repair type depends on the concerted presence of pathway specific damage response proteins [1, 2, 46–48]. The main two ways by which cells respond to DNA double-strand breaks are non-homologous end joining (NHEJ; also called canonical NHEJ = cNHEJ) and homologous recombination (HR). NHEJ mediated DSB repair is fast and can be error-prone, but it can be flexibly performed throughout all cell cycle phases. HR works error-free, but is mostly restricted to late S and G2 phases as a homologous sister chromatid is required as a repair template [49–52]. Recent data, however, have suggested that active genes may employ HR also in G1 phase, by utilizing the nascent RNA as a template for precise repair (reviewed in [53]). As the DNA-end resection is inhibited in G1 cells, an alternative model with cNHEJ taking the advantage of the same principle (RNA-templated repair) has also been proposed. Interestingly, DNA repair by HR is preferred in lower eukaryotic life forms, whereas NHEJ is predominantly observed

**3. DNA double strand break repair mechanisms**

**46**

After the induction of a DSB, damage response proteins are rapidly recruited and accurately determine the fate of the DSB towards a repair pathway that best deals with the damage site in a certain genomic and cellular context. The chromatin remodeling p53-binding protein (53BP1) protects the break site from extensive end resection [60], thereby promoting repair by non-homologous end joining [61], whereas BRCA1 facilitates extensive end resection for repair by homologous recombination [52, 62].

#### **3.2 Non-homologous end joining**

The NHEJ repair pathway is initiated with binding of the Ku70-Ku80 heterodimer complex to the DNA ends of the DSB site, which serves as a linkage between damage site and further damage response proteins [61, 63]. In a second step, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited to the Ku complex forming the DNA-PK complex. On-going recruitment of X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF to the DNA-PK complex forms the core NHEJ complex [64]. DNA-PKcs sterically protects the break site for repair and phosphorylates other repair proteins [65, 66] and H2AX [41]. Furthermore, DNA-PK auto-phosphorylation results in a conformational change of the core complex, thereby enabling DNA end processing by nucleases and dissociation of the DNA-PKcs subunit [67, 68]. Finally, ligation of the DNA ends is mediated by the X-ray complex (XRCC4)/DNA Ligase IV (X4LIG4) complex and XLF [69–71]. Artemis endonuclease [72, 73], polynucleotide kinase (PNK) [74], DNA polymerase (pol) μ and λ can be additionally involved in NHEJ repair depending on the chemical properties of the DNA damage site [75].

#### **3.3 Homologous recombination**

To initiate repair by HR, the free damaged DNA ends at the DSB site must be sensed and bound by a protein complex comprised of MRE11, RAD50 and NBS1 (MRN complex) [76]. Next, the protein kinase Ataxia Telangiectasia Mutated (ATM) [77] is recruited to the MRN complex at the damage site [78], which autophosphorylates and phosphorylates components of the neighboring chromatin. Most prominent phosphorylations are those of the histone variant H2AX (γH2AX), one of the earliest and a very sensitive marker of cellular response to DSBs [38]. End resection is initiated by the single-strand endonuclease and exonuclease activity of the Mre11 protein [52, 79] of the MRN complex. RAD50 further stimulates Mre11 nuclease activity and Nbs1 interacts with CtIP [80], another protein that is essential for the initiation of MRN complex mediated end resection [81]. Exonuclease 1 (Exo1) and Dna1/BLM are recruited by CtIP to continue end resection [82–84] until it gets attenuated by RPA coating of resected ssDNA ends [85]. BRCA2 in combination with BRCA1 and PALB2 dismantles the ssDNA ends from RPA coats enabling binding and forming of the RAD51 nucleoprotein filament, which stimulates

homology search and strand invasion [86]. Sister chromatid strand recombination via Holiday junctions is further facilitated by RAD54A and its paralog RAD54B [87, 88], finally resulting in conservative repair of the DNA lesion.

### **3.4 Alternative repair pathways**

a-NHEJ or b-NHEJ has been described in slightly different ways which are not well distinguished [56–58]. Mostly, in the presence of short micro-homologies (>4 bp) after CtIP-MRN mediated end resection, repair via an alternative end joining (MMEJ) can take place [89]. This is initiated by Poly(ADP-ribose) polymerase 1 (PARP1) and followed up by DNA polymerase θ (pol θ) mediated strand extension starting at the paired micro-homology site. Ligase1 and Ligase2 are supposed to perform the final ligation of DNA ends [90, 91].

When the damage site is flanked by larger regions with non-allelic sequence homologies, repair by single-strand annealing is also possible. The absence of Ku proteins and even more extensive end resection to expose the homologous regions as single strands are necessary for SSA repair [92]. Again, RAP binding to the resected ends promotes RAD52 mediated annealing of homologous regions. Nuclease XPF-ERCC1 trims the remaining non-homologous overhangs and DNA Ligase 1 connects the DNA ends [93].

Several studies indicate, that damaged genomic Alu elements use microhomologies for single-strand annealing, thereby often leading to translocations [94, 95]. Such nonconventional damage repair processes might explain a significant portion of the observed deletion events associated with malignancies [59]. In fact, in vitro model systems could already demonstrate Alu mediate non-allelic homology dependent DSB repair [96].
