Wael Y. Mansour

The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies

Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.

Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks.

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair

PURPOSE Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. MATERIALS & METHODS Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and gammaH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. RESULTS Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD(50) for 10 fractions 46.0Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25Gy]) and cell cultures (D(0.01); 7.1 vs. 3.5Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. CONCLUSIONS The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

Radiation-induced double-strand breaks require ATM but not Artemis for homologous recombination during S-phase

Double-strand breaks (DSBs) are repaired by two distinct pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). The endonuclease Artemis and the PIK kinase Ataxia-Telangiectasia Mutated (ATM), mutated in prominent human radiosensitivity syndromes, are essential for repairing a subset of DSBs via NHEJ in G1 and HR in G2. Both proteins have been implicated in DNA end resection, a mandatory step preceding homology search and strand pairing in HR. Here, we show that during S-phase Artemis but not ATM is dispensable for HR of radiation-induced DSBs. In replicating AT cells, numerous Rad51 foci form gradually, indicating a Rad51 recruitment process that is independent of ATM-mediated end resection. Those DSBs decorated with Rad51 persisted through S- and G2-phase indicating incomplete HR resulting in unrepaired DSBs and a pronounced G2 arrest. We demonstrate that in AT cells loading of Rad51 depends on functional ATR/Chk1. The ATR-dependent checkpoint response is most likely activated when the replication fork encounters radiation-induced single-strand breaks leading to generation of long stretches of single-stranded DNA. Together, these results provide new insight into the role of ATM for initiation and completion of HR during S- and G2-phase. The DSB repair defect during S-phase significantly contributes to the radiosensitivity of AT cells.

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.

Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3′-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

The CHD1 gene, encoding the chromo‐domain helicase DNA‐binding protein‐1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double‐strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR‐mediated DNA repair but not non‐homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.

Inhibition of PARP1‐dependent end‐joining contributes to Olaparib‐mediated radiosensitization in tumor cells

Poly‐ADP‐ribose‐polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication‐dependent and more profound in HR‐deficient tumors. Here, we present a new mode of PARPi‐mediated radiosensitization which was observed in four out of six HR‐proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication‐independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB‐repair is switched to PARP1‐dependent end‐joining (PARP1‐EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end‐joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1‐EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy.

Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer

Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome.

High levels of RAD51 perturb DNA replication elongation and cause unscheduled origin firing due to impaired CHK1 activation.

In response to replication stress ATR signaling through CHK1 controls the intra-S checkpoint and is required for the maintenance of genomic integrity. Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double strand breaks and interstrand crosslinks. In addition, HR, with its key player RAD51, provides critical support for the recovery of stalled forks during replication. High levels of RAD51 are regularly found in various cancers, yet little is known about the effect of the increased RAD51 expression on intra-S checkpoint signaling. Here, we describe a role for RAD51 in driving genomic instability caused by impaired replication and intra-S mediated CHK1 signaling by studying an inducible RAD51 overexpression model as well as 10 breast cancer cell lines. We demonstrate that an excess of RAD51 decreases I-Sce-I mediated HR despite formation of more RAD51 foci. Cells with high RAD51 levels display reduced elongation rates and excessive dormant origin firing during undisturbed growth and after damage, likely caused by impaired CHK1 activation. In consequence, the inability of cells with a surplus of RAD51 to properly repair complex DNA damage and to resolve replication stress leads to higher genomic instability and thus drives tumorigenesis.