Sites Of DNA Double-strand Breaks Research Articles

Dicer regulates non-homologous end joining and is associated with chemosensitivity in colon cancer patients.

DNA double-strand break (DSB) repair is an important mechanism underlying chemotherapy resistance in human cancers. Dicer participates in DSB repair by facilitating homologous recombination. However, whether Dicer is involved in non-homologous end joining (NHEJ) remains unknown. Here, we addressed whether Dicer regulates NHEJ and chemosensitivity in colon cancer cells. Using our recently developed NHEJ assay, we found that DSB introduction by I-SceI cleavage leads to Dicer upregulation. Dicer knockdown increased SIRT7 binding and decreased the level of H3K18Ac (acetylated lysine 18 of histone H3) at DSB sites, thereby repressing the recruitment of NHEJ factors to DSB sites and inhibiting NHEJ. Dicer overexpression reduced SIRT7 binding and increased the level of H3K18Ac at DSB sites, promoting the recruitment of NHEJ factors to DSBs and moderately enhancing NHEJ. Dicer knockdown and overexpression increased and decreased, respectively, the chemosensitivity of colon cancer cells. Dicer protein expression in colon cancer tissues of patients was directly correlated with chemoresistance. Our findings revealed a function of Dicer in NHEJ-mediated DSB repair and the association of Dicer expression with chemoresistance in colon cancer patients.

Abstract 2477: Genomic instability in BRCA1-deficient cells is a result of the anti-recombinogenic activity of BLM helicase

Abstract Mutations in BRCA1 are responsible for approximately 5% of cases of breast cancer, and there are few treatment options that substantially alter the probability of disease onset in individuals with BRCA1 mutations. BRCA1-deficient cells exhibit increased genomic instability following DNA damaging treatments due to a defect in the homologous recombination (HR) DNA repair pathway. Presently, Olaparib, a PARP inhibitor, is approved as a targeted monotherapy for germline BRCA1 mutated advanced ovarian cancers. As use of this treatment has expanded, data suggests that patients exhibit a further relapse and resistance to PARP inhibitors via mechanisms that include the development of secondary BRCA1 reversion mutations, enhanced drug efflux relating to P-glycoprotein, and mutations in other DNA repair proteins that restore HR. Here, we show that the RECQ helicase, BLM, mediates the genomic instability observed in BRCA1-deficient cells by a mechanism that depends in part on its anti-recombinogenic activity. We have generated conditional knockout mice with single and combined deficiencies in BRCA1, BLM, and 53BP1 in the B lymphocyte cell population. Ablation of BLM in BRCA1-deficient cells allows the HR repair pathway to be restored, leading to a partial rescue of the genomic instability that is present in BRCA1-deficient cells. The stable accumulation of RAD51, a marker for HR, at DNA double-strand break sites is inhibited by BLM in BRCA1-deficient B cells. However, DNA end resection is not impacted by single or co-deletion of BRCA1 and BLM. Furthermore, cells co-deficient in BRCA1 and BLM display limited sensitivity to PARP inhibition. The rescue in HR and PARPi sensitivity phenotypes following deletion of BLM is only present in hypomorphic BRCA1Δ11/Δ11 cells and not the RING domain deficient BRCA1Δ2/Δ2. These results demonstrate that the anti-recombinogenic activity of BLM is of potentially great significance for regulating the balance of HR versus other error-prone repair pathways. Mutation of BLM in BRCA1-deficient tumors is therefore a potential pathway leading to resistance to PARPi and other DNA damaging agents. Citation Format: Dharm S. Patel, Sarah M. Misenko, Samuel F. Bunting. Genomic instability in BRCA1-deficient cells is a result of the anti-recombinogenic activity of BLM helicase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2477. doi:10.1158/1538-7445.AM2017-2477

Abstract 2240: “Smart” selenium compound ISC-4 inhibits androgen receptor signaling and induces ROS- and p53-dependent apoptosis in LNCaP prostate cancer cells

Abstract “Smart” selenium compound phenylbutyl isoselenocyanate (ISC-4) was developed by substituting sulfur in cancer chemopreventive compound phenylbutyl isothiocyanate (PBITC) with selenium, resulting in increased anti-cancer activity in several melanoma and colon cancer cell lines. The in vivo anticancer beneficial effects of ISC-4 have been reported in both melanoma and colon cancer mouse models as well. To explore the potential chemopreventive attributes of ISC-4 against prostate carcinogenesis, we selected LNCaP cells (with wild type p53 and functional androgen receptor (AR) signaling) to examine the growth suppression and apoptosis responses in cell culture and interrogated signaling mechanisms through pharmacological and siRNA knockdown approaches. Results show that when compared with PBITC, ISC-4 was more potent at suppressing cell growth and inducing apoptotic cell death in LNCaP cells. ISC-4 induced caspase-mediated apoptosis (as evidenced by cleavage of PARP) in dose- and time-dependent manners in association with decreased AR and its best known target prostate specific antigen (PSA) protein abundance and upregulation of p53-DNA damage response (DDR) protein expression levels: p53, its canonical target p21, and p-H2AX that marks DNA double-strand break sites. Similar to the rapid induction of reactive oxygen species (ROS) by isothiocyanates, ISC-4 exposure resulted in increased dihydroethidium-detectable ROS in a concentration-dependent manner. Preincubation with the ROS scavenger N-Acetyl-L-cysteine (NAC, 10 µM to 2 mM), markedly reduced ISC-4-induced cell death and attenuated p53, p21 and p-H2AX. Moreover, siRNA knockdown of p53 did not suppress ROS production, but decreased ISC-4-induced Annexin V-positive apoptosis, PARP cleavage and the DDR proteins. Taken together, these data suggest that ISC-4 inhibits LNCaP cell growth and survival with concomitant suppression of AR signaling and induction of ROS-p53-DDR mediated apoptosis. Given the pivotal significance of AR signaling in prostate epithelial survival, cancer genesis and progression, and the preservation of wild type p53 in precancerous lesions and early stage prostate cancer, our findings suggest attractive chemopreventive potential of ISC-4 against prostate carcinogenesis. Citation Format: Wei Wu, Kartick Pramanik, Cheng Jiang, Shantu Amin, Arun K. Sharma, Junxuan Lu. “Smart” selenium compound ISC-4 inhibits androgen receptor signaling and induces ROS- and p53-dependent apoptosis in LNCaP prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2240. doi:10.1158/1538-7445.AM2017-2240

Nuclear TRADD prevents DNA damage-mediated death by facilitating non-homologous end-joining repair

TNF receptor-associated death domain (TRADD) is an essential mediator of TNF receptor signaling, and serves as an adaptor to recruit other effectors. TRADD has been shown to cycle between the cytoplasm and nucleus due to its nuclear localization (NLS) and export sequences (NES). However, the underlying function of nuclear TRADD is poorly understood. Here we demonstrate that cytoplasmic TRADD translocates to DNA double-strand break sites (DSBs) during the DNA damage response (DDR). Deficiency of TRADD or its sequestration in cytosol leads to accumulation of γH2AX-positive foci in response to DNA damage, which is reversed by nuclear TRADD expression. TRADD facilitates non-homologous end-joining (NHEJ) by recruiting NHEJ repair factors 53BP1 and Ku70/80 complex, whereas TRADD is dispensable for homologous recombination (HR) repair. Finally, an impaired nuclear localization of TRADD triggers cell death through the persistent activation of JNK and accumulation of reactive oxygen species (ROS). Thus, our findings suggest that translocation of TRADD to DSBs into the nucleus contributes to cell survival in response to DNA damage through an activation of DNA damage repair.

Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair.

Upon DNA damage, histone modifications are dynamically reshaped to accommodate DNA damage signaling and repair within chromatin. In this study, we report the identification of the histone demethylase KDM5A as a key regulator of the bromodomain protein ZMYND8 and NuRD (nucleosome remodeling and histone deacetylation) complex in the DNA damage response. We observe KDM5A-dependent H3K4me3 demethylation within chromatin near DNA double-strand break (DSB) sites. Mechanistically, demethylation of H3K4me3 is required for ZMYND8-NuRD binding to chromatin and recruitment to DNA damage. Functionally, KDM5A deficiency results in impaired transcriptional silencing and repair of DSBs by homologous recombination. Thus, this study identifies a crucial function for KDM5A in demethylating H3K4 to allow ZMYND8-NuRD to operate within damaged chromatin to repair DSBs.

Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks

A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination.

Abstract B45: A genome-scale screen identifies the microcephaly gene, ZNF335, as a regulator of DNA end resection

Abstract Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair pathway that is of critical importance during the S and G2 phases of the mammalian cell cycle. The limiting step in the HR pathway is the generation of single-stranded DNA (ssDNA) by resection of DNA ends at the DSB site. We have established a high-throughput immunofluorescence-based assay that monitors DNA end resection by plate-based laser scanning cytometry. Employing this assay we screened a genome-scale siRNA library that targeted approximately 18,500 genes. Remarkably, the top hits in our screen were known regulators of resection including CtIP and all three subunits of the MRN complex. I will present ongoing work on one of the hits from the screen, the zinc finger protein ZNF335. The ZNF335 gene was recently found to be mutated in a syndrome that causes some of the worst cases of neonatal microcephaly ever observed. Numerous genome instability syndromes have been documented to display microcephaly as a clinical feature, including Seckel syndrome, which can be caused by mutations in the known resection genes CtIP and DNA2. We first validated the results of the screen by demonstrating that ZNF335 promotes resection and repair by HR. We determined that the four C-terminal zinc finger domains of ZNF335 are necessary and sufficient for its function in resection. Depletion of ZNF335 inhibits phosphorylation of CHK1 at a characterized ATR consensus site, suggesting that ZNF335 is needed for activation of ATR following a DSB. We examined the recruitment of known resection factors to DSB sites and determined that the localization of CtIP and BLM were impaired in cells depleted of ZNF335. Our data suggests that ZNF335 plays an important role in promoting resection and ATR activation in response to DSBs. Citation Format: Jordan Young, Mikhail Bashkurov, Andrea McEwan, Thomas Sun, Alessandro Datti, Daniel Durocher. A genome-scale screen identifies the microcephaly gene, ZNF335, as a regulator of DNA end resection [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B45.

Regulatory cross-talk determines the cellular levels of 53BP1 protein, a critical factor in DNA repair

DNA double strand breaks (DSBs) severely disrupt DNA integrity. 53BP1 plays critical roles in determining DSB repair. Whereas the recruitment of 53BP1 to the DSB site is key for its function, recent evidence suggests that 53BP1's abundance also plays an important role in DSB repair because recruitment to damage sites will be influenced by protein availability. Initial evidence has pointed to three proteins, the ubiquitin-conjugating enzyme UbcH7, the cysteine protease cathepsin L (CTSL), and the nuclear structure protein lamin A/C, that may impact 53BP1 levels, but the roles of each protein and any interplay between them were unclear. Here we report that UbcH7-dependent degradation plays a major role in controlling 53BP1 levels both under normal growth conditions and during DNA damage. CTSL influenced 53BP1 degradation during DNA damage while having little effect under normal growth conditions. Interestingly, both the protein and the mRNA levels of CTSL were reduced in UbcH7-depleted cells. Lamin A/C interacted with 53BP1 under normal conditions. DNA damage disrupted the lamin A/C-53BP1 interaction, which preceded the degradation of 53BP1 in soluble, but not chromatin-enriched, cellular fractions. Inhibition of 53BP1 degradation by a proteasome inhibitor or by UbcH7 depletion restored the 53BP1-lamin A/C interaction. Depletion of lamin A/C, but not CTSL, caused a similar enhancement in cell sensitivity to DNA damage as UbcH7 depletion. These data suggest that multiple pathways collectively fine-tune the cellular levels of 53BP1 protein to ensure proper DSB repair and cell survival.

Abstract IA27: Exploiting the inhibition of cullin-RING-ligases in DSB repair as a therapeutic strategy

Abstract While single component E3 ubiquitin ligases have established roles in DNA double-strand break (DSB) repair, the function of multicomponent cullin-RING-ligases (CRL) in DSB repair is only beginning to emerge. FBXW7 is a substrate recognition component of Skp1-Cullin1-F-box E3 ubiquitin ligases previously known only to regulate the proteasomal degradation of substrates such as Cyclin E and MCL1. Given that FBXW7 loss promotes genomic instability, we hypothesized that FBXW7 may have a direct function in DSB repair. To establish the functions of FBXW7 in DSB repair, we first demonstrated the rapid localization of FBXW7 to DSB sites in an ATM-dependent manner. Subsequently, we found that FBXW7 depletion impaired nonhomologous end-joining (NHEJ), but not homologous recombination (HR) repair, resulting in persistent radiation-induced DSBs. Investigation of the molecular mechanisms of FBXW7 in NHEJ revealed that FBXW7 interacts with and promotes K63-linked ubiquitination of XRCC4. This ubiquitination of XRCC4 promotes interaction between the XRCC4/XLF/LIG4 and DNAPK/KU70/KU80 complexes to facilitate NHEJ. To begin to therapeutically leverage this mechanism, strategies to both pharmacologically inhibit FBXW7-CRLs and to exploit FBXW7 mutations occurring in human cancers are being developed. To address the former, pevonedistat (MLN4924), an agent which inhibits CRLs via inhibition of cullin-neddylation, inhibits FBXW7-mediated XRCC4 ubiquitination and NHEJ. This activity leads to increased sensitivity of tumor cells to chemotherapy and radiation and represents a strategy that may be particularly effective in cancers with other DSB repair defects. As another strategy to leverage the mechanisms of FBXW7 in NHEJ, loss-of-function mutations in the WD domain of FBXW7 which occur frequently in human cancers are being explored. These mutations render cells defective in NHEJ and more sensitive to DNA damage. Thus, we hypothesize that inhibition of other DSB repair pathways (e.g. alternative-end-joining) may be an effective therapeutic strategy for FBXW7 mutant cancers. These novel mechanisms of NHEJ regulation as well as their implications in the treatment of human cancers with an emphasis on pancreatic and colorectal cancers will be discussed. This work was supported by NIH grants R01CA163895, P50CA130810, R01CA118762, R01CA156744, and R01CA171277. Citation Format: Meredith Morgan. Exploiting the inhibition of cullin-RING-ligases in DSB repair as a therapeutic strategy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr IA27.

Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy

Beclin 1 is a well-established core mammalian autophagy protein that is embryonically indispensable and has been presumed to suppress oncogenesis via an autophagy-mediated mechanism. Here, we show that Beclin 1 is a prenatal primary cytoplasmic protein but rapidly relocated into the nucleus during postnatal development in mice. Surprisingly, deletion of beclin1 in in vitro human cells did not block an autophagy response, but attenuated the expression of several DNA double-strand break (DSB) repair proteins and formation of repair complexes, and reduced an ability to repair DNA in the cells exposed to ionizing radiation (IR). Overexpressing Beclin 1 improved the repair of IR-induced DSB, but did not restore an autophagy response in cells lacking autophagy gene Atg7, suggesting that Beclin 1 may regulate DSB repair independent of autophagy in the cells exposed to IR. Indeed, we found that Beclin 1 could directly interact with DNA topoisomerase IIβ and was recruited to the DSB sites by the interaction. These findings reveal a novel function of Beclin 1 in regulation of DNA damage repair independent of its role in autophagy particularly when the cells are under radiation insult.

Increase of DNA damage and alteration of the DNA damage response in myelodysplastic syndromes and acute myeloid leukemias

Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We used immunofluorescence staining of γH2AX and 53BP1 for analyzing DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of the DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. As compared to γH2AX foci levels in normal bone marrow samples (0.2 focus per CD34+ cell±0.0; mean±standard error of mean), increased levels of γH2AX foci were detected in 16/16 MDS bone marrow samples (2.8 foci per CD34+ cell±0.5), 18/18 AML bone marrow samples (5.5 foci per blast±0.5), 1/1 MDS cell line (6.4 foci per cell) and 6/6 AML cell lines (12.0 foci per cell±0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. Further, DDR proteins were expressed heterogeneously suggesting impairment of the DDR. In summary, our results provide evidence for a continuous increase of DSB across the spectrum from MDS to AML in conjunction with an impaired DDR. Co-localization of γH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, γH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML.

Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells.

Interaction between RNF8 and DYRK2 is required for the recruitment of DNA repair molecules to DNA double-strand breaks.

The genome of eukaryotic cells is frequently exposed to damage by various genotoxins. Phosphorylation of histone H2AX at Serine 139 (γ-H2AX) is a hallmark of DNA damage. RNF8 monoubiquitinates γ-H2AX with the Lys63-linked ubiquitin chain to tether DNA repair molecules at DNA lesions. A high-throughput screening identified RNF8 as a binding partner of dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2). Notably, DNA damage-induced monoubiquitination of γ-H2AX is impaired in DYRK2-depleted cells. The foci formation of p53-binding protein 1 at DNA double-strand break sites is suppressed in DYRK2 knockdown cells, which fail to repair the DNA damage. A homologous recombination assay showed decreased repair efficiency in DYRK2-depleted cells. Our findings indicate direct interaction of DYRK2 with RNF8 in regulating response to DNA damage.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots.

Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined "hot spots." In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots.

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination

SummaryCanonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Imaging of Fluorescently Tagged ATM Kinase at the Sites of DNA Double Strand Breaks.

The combination of live cell imaging and laser micro-irradiation is an important technique to investigate the recruitment and kinetics of DNA repair molecules to DNA damage sites. In this chapter, we describe the detailed methods to study the dynamics of fluorescently tagged ATM protein kinase at laser-induced DNA double strand break (DSB) sites. The same protocol can be applied to analyze the recruitment and kinetics of other potential or known DNA repair proteins to DSB sites.

Cloning, localization and focus formation at DNA damage sites of canine Ku70

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.

And-1 is required for homologous recombination repair by regulating DNA end resection.

Homologous recombination (HR) is a major mechanism to repair DNA double-strand breaks (DSBs). Although tumor suppressor CtIP is critical for DSB end resection, a key initial event of HR repair, the mechanism regulating the recruitment of CtIP to DSB sites remains largely unknown. Here, we show that acidic nucleoplasmic DNA‐binding protein 1 (And‐1) forms complexes with CtIP as well as other repair proteins, and is essential for HR repair by regulating DSB end resection. Furthermore, And-1 is recruited to DNA DSB sites in a manner dependent on MDC1, BRCA1 and ATM, down-regulation of And-1 impairs end resection by reducing the recruitment of CtIP to damage sites, and considerably reduces Chk1 activation and other damage response during HR repair. These findings collectively demonstrate a hitherto unknown role of MDC1→And-1→CtIP axis that regulates CtIP-mediated DNA end resection and cellular response to DSBs.

Novel Role of LMO2 in DNA Repair Control in Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of Non-Hodgkin Lymphoma (NHL). However, about 40% of patients still do not respond to the current therapy and die. Therefore, there is an urgent need for developing new strategies to improve their outcome. We have shown that LIM domain Only 2 (LMO2) expression is increased in germinal center (GC) B-lymphocytes and GC-derived lymphomas. LMO2 protein is the best single prognostic biomarker which high expression is associated with longer survival of DLBCL patients treated with R-CHOP therapy, irrespective of the cell of origin of these tumors. LMO2 is indirectly involved in gene regulation by forming multipartite DNA-binding complexes with other transcription factors. LMO2 plays an important role in the normal development of hematopoietic and endothelial system, in erythropoiesis, and functions as an oncogene in T-cell acute lymphoblastic leukemia. However, its role in B cell biology as well as B-cell lymphoma is still unclear. We have recently shown that LMO2 over-expression in DLBCL results in genomic instability (Cubedo et al Blood 2012). These studies suggested that LMO2 expression could compromise proper DNA repair.To address whether LMO2 has a role in DNA repair, we evaluated the capacity of DLBCL cells with high expression of LMO2 (DLBCLLMO2+) to repair DNA double-strand breaks (DSBs). DNA DSBs are repaired by two major pathways: Non-homologous end joining (NHEJ) and Homologous recombination (HR). Therefore, next we evaluated whether LMO2 affects the activity of these DSBs repair pathways. To this end, we analyzed the core factors: 53BP1 for NHEJ and Rad51 for HR. We exposed DLBCLLMO2+ and controlcells to genotoxic agents used in DLBCL therapy to produce DSBs and evaluated 53BP1 and Rad51 recruitment to DSBs site via immunofluorescence (IF) studies. Phosphorylated form of H2AX, known as gammaH2AX was used as a DSB molecular marker. In these studies we found a similar frequency of 53BP1 foci in both DLBCLLMO2+ and control cells. These results suggested that NHEJ is active irrespective of LMO2 protein levels. However, DLBCLLMO2+ cells showed a decreased frequency of Rad51 foci after DNA damage. Importantly, DLBCLLMO2+ cells showed similar Rad51 protein levels and cell-cycle distribution when compared to control cells. These results showed a deficient HR pathway in DLBCLLMO2+ cells and suggested a role for LMO2 in the control of HR. In support of this, overexpression of LMO2 in control cells produced a similar HR-deficiency as observed in DLBCLLMO2+ cells. In addition, knockdown of LMO2 in DLBCLLMO2+ cells restored Rad51 foci formation to the levels observed in control cells. These results showed that HR activity in DLBCL cells depends on LMO2 protein levels. It is well known that deficiency in HR can be rescued by deleting 53BP1, which is a HR inhibitor. Hence, in order to address the question whether deletion of 53BP1 in DLBCLLMO2+ cells can rescue HR pathway, we knockdown 53BP1 in DLBCLLMO2+ cells via shRNA and examined the frequency of Rad51 foci after exposure to different genotoxic agents. Interestingly, we found an increase in Rad51 foci formation in DLBCLLMO2+sh53BP1 cells to near control cells levels. This result shows that LMO2-dependent inhibition of HR depends on 53BP1 and suggests that LMO2 and 53BP1 form a complex at the DSB site. Two observations support this hypothesis: 1) LMO2 co-localize with 53BP1 at the DSB site; and 2) LMO2 expression increases the frequency of 53BP1-Rif1 complex at the DSB site. Since Rif1 (Rap1-interacting factor 1) is one of the 53BP1 effector proteins that inhibits HR, here we propose that LMO2 inhibition of HR is through the stabilization of the 53BP1-Rif1 complex.Our study reveals a novel role for LMO2 in the control of genome stability by controlling homologous recombination repair activity. Defect in HR pathway in LMO2 expressing cells could explains why DLBCL patients with higher level of LMO2 are more sensitive to chemotherapy, and its significance as the best prognostic marker for survival. Our studies can potentially give new insight for the treatment of DLBCL patient and improve their outcome. DisclosuresNo relevant conflicts of interest to declare.