Characteristics of replication-independent endogenous double-strand breaks in Saccharomyces cerevisiae

Background

Replication-independent endogenous double-strand breaks (RIND-EDSBs) occur in both humans and yeast in the absence of inductive agents and DNA replication. In human cells, RIND-EDSBs are hypermethylated, preferentially retained in the heterochromatin and unbound by γ-H2AX. In single gene deletion yeast strains, the RIND-EDSB levels are altered; the number of RIND-EDSBs is higher in strains with deletions of histone deacetylase, endonucleases, topoisomerase, or DNA repair regulators, but lower in strains with deletions of the high-mobility group box proteins or Sir2. In summary, RIND-EDSBs are different from pathologic DSBs in terms of their causes and consequences. In this study, we identified the nucleotide sequences surrounding RIND-EDSBs and investigated the features of these sequences as well as their break locations.

Results

In recent work, we detected RIND-EDSBs using ligation mediated PCR. In this study, we sequenced RIND-EDSB PCR products of resting state Saccharomyces cerevisiae using next-generation sequencing to analyze RIND-EDSB sequences. We found that the break locations are scattered across a number of chromosomes. The number of breaks correlated with the size of the chromosomes. Most importantly, the break occurrences had sequence pattern specificity. Specifically, the majority of the breaks occurred immediately after the sequence “ACGT” (P = 2.2E-156). Because the “ACGT” sequence does not occur primarily in the yeast genome, this specificity of the “ACGT” sequence cannot be attributed to chance.

Conclusions

RIND-EDSBs occur non-randomly; that is, they are produced and retained by specific mechanisms. Because these particular mechanisms regulate their generation and they possess potentially specific functions, RIND-EDSBs could be epigenetic marks.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-750) contains supplementary material, which is available to authorized users.     

LINE-1 methylation status of endogenous DNA double-strand breaks

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.     

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.     

The requirement of Artemis in double-strand break repair depends on the type of DNA damage

Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.     

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.     

Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.     

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.     

Differential Requirement for SUB1 in Chromosomal and Plasmid Double-Strand DNA Break Repair

Non homologous end joining (NHEJ) is an important process that repairs double strand DNA breaks (DSBs) in eukaryotic cells. Cells defective in NHEJ are unable to join chromosomal breaks. Two different NHEJ assays are typically used to determine the efficiency of NHEJ. One requires NHEJ of linearized plasmid DNA transformed into the test organism; the other requires NHEJ of a single chromosomal break induced either by HO endonuclease or the I-SceI restriction enzyme. These two assays are generally considered equivalent and rely on the same set of NHEJ genes. PC4 is an abundant DNA binding protein that has been suggested to stimulate NHEJ. Here we tested the role of PC4''s yeast homolog SUB1 in repair of DNA double strand breaks using different assays. We found SUB1 is required for NHEJ repair of DSBs in plasmid DNA, but not in chromosomal DNA. Our results suggest that these two assays, while similar are not equivalent and that repair of plasmid DNA requires additional factor(s) that are not required for NHEJ repair of chromosomal double-strand DNA breaks. Possible roles for Sub1 proteins in NHEJ of plasmid DNA are discussed.     

Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.     

The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining

Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of γ-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.     

TDP1 is required for efficient non-homologous end joining in human cells

Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3′ and 5′ terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.     

Saccharomyces cerevisiae DNA Ligase IV Supports Imprecise End Joining Independently of Its Catalytic Activity

DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5′ resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.     

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.     

DNA Damage Signalling and Repair in Dictyostelium discoideum

Repair of DNA double strand breaks (DSBs) is critical for the maintenance of genome integrity. DNA DSBs can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). Whilst HR requires sequences homologous to thedamaged DNA template in order to facilitate repair, NHEJ occurs through recognition of DNA DSBs by a variety of proteins that process and rejoin DNA termini by direct ligation. Here we review two recent reports that NHEJ is conserved in the social amoebaDictyostelium discoideum. Certain components of the mammalian NHEJ pathway that are absent in genetically tractable organisms such as yeast are present in Dictyostelium and we discuss potential directions for future research, in addition to considering this organism as a genetic model system for the study of NHEJ in vivo.     

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.     

HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

Background

Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood.

Results

Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway.

Conclusions

This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.     

Increased Gene Targeting in Ku70 and Xrcc4 Transiently Deficient Human Somatic Cells

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell’s genome is important to advance strategies for biotechnology and genetic medicine.     

Non-homologous DNA end joining

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.     

Regulation of DNA double-strand break repair pathway choice

DNA double-strand breaks （DSBs） are critical lesions that can result in cell death or a wide variety of genetic alterations including largeor small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining （NHEJ） and homologous recombination （HR）, and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1（XRS2） complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit （DNA-PKcs）, and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.