Factors determining DNA double-strand break repair pathway choice in G2 phase

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.     

Molecular basis for DNA strand displacement by NHEJ repair polymerases

The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair.     

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism.     

Pathways for Double-strand Break Repair in Genetically Unstable Z-DNA-forming Sequences

DNA can adopt many structures that differ from the canonical B-form, and several of these non-canonical DNA structures have been implicated in genetic instability associated with human disease. Earlier, we found that Z-DNA causes DNA double-strand breaks (DSBs) in mammalian cells that can result in large-scale deletions and rearrangements. In contrast, the same Z-DNA-forming CG repeat in Escherichia coli resulted in only small contractions or expansions within the repeat. This difference in the Z-DNA-induced mutation spectrum between mammals and bacteria might be due to different mechanisms for DSB repair; in mammalian cells, non-homologous end-joining (NHEJ) is a major DSB repair pathway, while E. coli do not contain this system and typically use homologous recombination (HR) to process DSBs. To test the extent to which the different DSB repair pathways influenced the Z-DNA-induced mutagenesis, we engineered bacterial E.coli strains to express an inducible NHEJ system, to mimic the situation in mammalian cells. Mycobacterium tuberculosis NHEJ proteins Ku and ligase D (LigD) were expressed in E.coli cells in the presence or absence of HR, and the Z-DNA-induced mutations were characterized. We found that the presence of the NHEJ mechanism markedly shifted the mutation spectrum from small deletions/insertions to large-scale deletions (from 2% to 24%). Our results demonstrate that NHEJ plays a role in the generation of Z-DNA-induced large-scale deletions, suggesting that this pathway is associated with DNA structure-induced destabilization of genomes from prokaryotes to eukaryotes.     

Overhang polarity of chromosomal double-strand breaks impacts kinetics and fidelity of yeast non-homologous end joining

Non-homologous end joining (NHEJ) is the main repair pathway for DNA double-strand breaks (DSBs) in cells with limited 5′ resection. To better understand how overhang polarity of chromosomal DSBs affects NHEJ, we made site-specific 5′-overhanging DSBs (5′ DSBs) in yeast using an optimized zinc finger nuclease at an efficiency that approached HO-induced 3′ DSB formation. When controlled for the extent of DSB formation, repair monitoring suggested that chromosomal 5′ DSBs were rejoined more efficiently than 3′ DSBs, consistent with a robust recruitment of NHEJ proteins to 5′ DSBs. Ligation-mediated qPCR revealed that Mre11-Rad50-Xrs2 rapidly modified 5′ DSBs and facilitated protection of 3′ DSBs, likely through recognition of overhang polarity by the Mre11 nuclease. Next-generation sequencing revealed that NHEJ at 5′ DSBs had a higher mutation frequency, and validated the differential requirement of Pol4 polymerase at 3′ and 5′ DSBs. The end processing enzyme Tdp1 did not impact joining fidelity at chromosomal 5′ DSBs as in previous plasmid studies, although Tdp1 was recruited to only 5′ DSBs in a Ku-independent manner. These results suggest distinct DSB handling based on overhang polarity that impacts NHEJ kinetics and fidelity through differential recruitment and action of DSB modifying enzymes.     

Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks

Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G₁ cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5′ to 3′ resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G₁, and markedly repressed at G₂. Repression of NHEJ at G₂ is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5′ end resection by CDK1 inhibition at G₂ alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G₁. Expression of excess Ku can partially offset the inhibition of end joining at G₂. The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.     

New Insights into NHEJ Repair Processes in Prokaryotes

In eukaryotic cells, the repair of DNA double strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway is critical for genome stability. Until recently it was assumed that this DSB repair pathway was restricted to the eukarya. However, a functionally homologous prokaryotic NHEJ repair apparatus has now been identified and characterised. In contrast to the complex eukaryotic system, bacterial NHEJ appears to require only two proteins, Ku and a multifunctional DNA ligase, which form a two-component repair complex at the termini of DSBs. Together, these DNA repair factors possess all of the break-recognition, end-processing and ligation activities required to facilitate the complex task of DSB repair, both in vitro and in vivo. Our recent findings lay the foundation for understanding the molecular mechanisms that co-ordinate the processing and joining of DSBs by NHEJ in bacteria and also provides a conceptual framework for delineating the end-processing reactions in eukaryotes.     

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo.     

Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma: Implications for Genome Stability

Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since both impaired and hyperactive double strand break (DSB) repair pathways can result in DNA rearrangements, we investigated the functionality of DSB repair in MM cells. Repair kinetics of ionizing-radiation (IR)-induced DSBs was similar in MM and normal control lymphoblastoid cell lines, as revealed by the comet assay. However, four out of seven MM cell lines analyzed exhibited a subset of persistent DSBs, marked by γ-H2AX and Rad51 foci that elicited a prolonged G2/M DNA damage checkpoint activation and hypersensitivity to IR, especially in the presence of checkpoint inhibitors. An analysis of the proteins involved in DSB repair in MM cells revealed upregulation of DNA-PKcs, Artemis and XRCC4, that participate in non-homologous end joining (NHEJ), and Rad51, involved in homologous recombination (HR). Accordingly, activity of both NHEJ and HR were elevated in MM cells compared to controls, as determined by in vivo functional assays. Interestingly, levels of proteins involved in a highly mutagenic, translocation-promoting, alternative NHEJ subpathway (Alt-NHEJ) were also increased in all MM cell lines, with the Alt-NHEJ protein DNA ligase IIIα, also overexpressed in several plasma cell samples isolated from MM patients. Overactivation of the Alt-NHEJ pathway was revealed in MM cells by larger deletions and higher sequence microhomology at repair junctions, which were reduced by chemical inhibition of the pathway. Taken together, our results uncover a deregulated DSB repair in MM that might underlie the characteristic genome instability of the disease, and could be therapeutically exploited.     

Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae

The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway. NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences. Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA. Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group. Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes. Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed.     

Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

High-linear energy transfer ionizing radiation, derived from high charge (Z) and energy (E) (HZE) particles, induces clustered/complex DNA double-strand breaks (DSBs) that include small DNA fragments, which are not repaired by the non-homologous end-joining (NHEJ) pathway. The homologous recombination (HR) DNA repair pathway plays a major role in repairing DSBs induced by HZE particles. The Mre11 complex (Mre11/Rad50/NBS1)-mediated resection of DSB ends is a required step in preparing for DSB repair via the HR DNA repair pathway. Here we found that expression of Bcl2 results in decreased HR activity and retards the repair of DSBs induced by HZE particles (i.e. ⁵⁶iron and ²⁸silicon) by inhibiting Mre11 complex activity. Exposure of cells to ⁵⁶iron or ²⁸silicon promotes Bcl2 to interact with Mre11 via the BH1 and BH4 domains. Purified Bcl2 protein directly suppresses Mre11 complex-mediated DNA resection in vitro. Expression of Bcl2 reduces the ability of Mre11 to bind DNA following exposure of cells to HZE particles. Our findings suggest that, after cellular exposure to HZE particles, Bcl2 may inhibit Mre11 complex-mediated DNA resection leading to suppression of the HR-mediated DSB repair in surviving cells, which may potentially contribute to tumor development.     

Repair of a minimal DNA double-strand break by NHEJ requires DNA-PKcs and is controlled by the ATM/ATR checkpoint

Mammalian cells primarily rejoin DNA double-strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway. The joining of the broken DNA ends appears directly without template and accuracy is ensured by the NHEJ factors that are under ATM/ATR regulated checkpoint control. In the current study we report the engineering of a mono-specific DNA damaging agent. This was used to study the molecular requirements for the repair of the least complex DSB in vivo. Single-chain PvuII restriction enzymes fused to protein delivery sequences transduce cells efficiently and induce blunt end DSBs in vivo. We demonstrate that beside XRCC4/LigaseIV and KU, the DNA-PK catalytic subunit (DNA-PKcs) is also essential for the joining of this low complex DSB in vivo. The appearance of blunt end 3′-hydroxyl and 5′-phosphate DNA DSBs induces a significantly higher frequency of anaphase bridges in cells that do not contain functional DNA-PKcs, suggesting an absolute requirement for DNA-PKcs in the control of chromosomal stability during end joining. Moreover, these minimal blunt end DSBs are sufficient to induce a p53 and ATM/ATR checkpoint function.     

Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR.     

Interacting proteins Rtt109 and Vps75 affect the efficiency of non-homologous end-joining in Saccharomyces cerevisiae

One of the key pathways for DNA double-stranded break (DSB) repair is the non-homologous end-joining (NHEJ) pathway, which directly re-ligates two broken ends of DNA. Using a plasmid repair assay screen, we identified that the deletion strain for RTT109 had a reduced efficiency for NHEJ in yeast. This deletion strain also had a reduced efficiency to repair induced chromosomal DSBs in vivo. Tandem-affinity purification of Rtt109 recovered Vps75 as a physical interacting protein. Deletion of VPS75 was also shown to have an effect on the efficiency of NHEJ in both the plasmid repair and the chromosomal repair assays. In addition, deletion mutants for both RTT109 and VPS75 showed hypersensitivity to different DNA damaging agents. Our genetic interaction analysis supports a role for RTT109 in DNA damage repair. We propose that one function of the Rtt109-Vps75 interacting protein pair is to affect the efficiency of NHEJ in yeast. Vps75 but not Rtt109 also seem to have an effect on the efficiency of DSB repair using homologous recombination.     

Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr⁵⁴⁰ amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.     

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.     

DNA-PK: A dynamic enzyme in a versatile DSB repair pathway

DNA double stranded breaks (DSBs) are the most cytoxic DNA lesion as the inability to properly repair them can lead to genomic instability and tumorigenesis. The prominent DSB repair pathway in humans is non-homologous end-joining (NHEJ). In the simplest sense, NHEJ mediates the direct re-ligation of the broken DNA molecule. However, NHEJ is a complex and versatile process that can repair DSBs with a variety of damages and ends via the utilization of a significant number of proteins. In this review we will describe the important factors and mechanisms modulating NHEJ with emphasis given to the versatility of this repair process and the DNA-PK complex.     

The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs. To address this, we have induced DSBs in fluorescently tagged mammalian cells (Ku80-EGFP, DNA-PKcs-YFP or XRCC4-GFP, key proteins in NHEJ) using ultra-soft X-rays (USX) or multi-photon near infrared (NIR) laser irradiation. We have shown in real-time that simple DSBs, induced by USX or NIR microbeam irradiation, are repaired rapidly involving Ku70/80 and XRCC4/Ligase IV/XLF. In contrast, DSBs with greater chemical complexity are repaired slowly involving not only Ku70/80 and XRCC4/Ligase IV/XLF but also DNA-PKcs. Ataxia telangiectasia-mutated inhibition only retards repair of the more chemically complex DSBs which require DNA-PKcs. In summary, the repair of DSBs by NHEJ is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB.