The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand... Show moreThe BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other highrisk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of Show less
The sensitivity to PARP inhibitors (PARPi) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. A robust method to identify HR... Show moreThe sensitivity to PARP inhibitors (PARPi) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2-related deficiencies. A robust method to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we evaluated the use of a functional test (the RECAP test) for the identification of HRD ovarian carcinomas. Forty-nine epithelial ovarian carcinomas (EOC) were analyzed by the RECAP test. Thirty-nine of these tumors were of the high-grade serous (HGSOC) histologic subtype. Ten out of these 39 HGSOC specimens showed HRD (26%), whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Eight out of 9 sequenced HRD tumors showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation. This study shows that the RECAP test is a reliable and rapid test to identify functional deficiencies in HR and a good alternative to DNA-based HRD tests.Recent studies have shown that the efficacy of PARP inhibitors in epithelial ovarian carcinoma (EOC) is related to tumor-specific defects in homologous recombination (HR) and extends beyond BRCA1/2 deficient EOC. A robust method with which to identify HR-deficient (HRD) carcinomas is therefore of utmost clinical importance. In this study, we investigated the proficiency of a functional HR assay based on the detection of RAD51 foci, the REcombination CAPacity (RECAP) test, in identifying HRD tumors in a cohort of prospectively collected epithelial ovarian carcinomas (EOCs). Of the 39 high-grade serous ovarian carcinomas (HGSOC), the RECAP test detected 26% (10/39) to be HRD, whereas ovarian carcinomas of other histologic subtypes (n = 10) were all HR-proficient (HRP). Of the HRD tumors that could be sequenced, 8/9 showed pathogenic BRCA1/2 variants or BRCA1 promoter hypermethylation, indicating that the RECAP test reliably identifies HRD, including but not limited to tumors related to BRCA1/2 deficiency. Furthermore, we found a trend towards better overall survival (OS) of HGSOC patients with RECAP-identified HRD tumors compared to patients with HRP tumors. This study shows that the RECAP test is an attractive alternative to DNA-based HRD tests, and further development of a clinical grade RECAP test is clearly warranted. Show less
The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to... Show moreThe Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in Forster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5 ' extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends. Show less
Zelensky, A.N.; Schoonakker, M.; Brandsma, I.; Tijsterman, M.; Gent, D.C. van; Essers, J.; Kanaar, R. 2020
Extrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double... Show moreExtrachromosomal DNA can integrate into the genome with no sequence specificity producing an insertional mutation. This process, which is referred to as random integration (RI), requires a double stranded break (DSB) in the genome. Inducing DSBs by various means, including ionizing radiation, increases the frequency of integration. Here we report that non-lethal physiologically relevant doses of ionizing radiation (10-100 mGy), within the range produced by medical imaging equipment, stimulate RI of transfected and viral episomal DNA in human and mouse cells with an extremely high efficiency. Genetic analysis of the stimulated RI (S-RI) revealed that it is distinct from the background RI, requires histone H2AX S139 phosphorylation (gamma H2AX) and is not reduced by DNA polymerase theta (Polq) inactivation. S-RI efficiency was unaffected by the main DSB repair pathway (homologous recombination and non-homologous end joining) disruptions, but double deficiency in MDC1 and 53BP1 phenocopies gamma H2AX inactivation. The robust responsiveness of S-RI to physiological amounts of DSBs can be exploited for extremely sensitive, macroscopic and direct detection of DSB-induced mutations, and warrants further exploration in vivo to determine if the phenomenon has implications for radiation risk assessment.Author summaryNot all DNA in mammalian nuclei is organized into chromosomes. The pool of extrachromosomal DNA molecules is produced by natural process: during genomic DNA repair, viral infections, phagocytosis; and in experimental settings after transfection, and in gene therapy. Extrachromosomal DNA can integrate into the genome at the site of a double-stranded DNA break (DSB), producing a mutation in the chromosome. Because DSBs are dangerous lesions, they are actively eliminated, and their availability is a limiting factor for extrachromosomal DNA integration. It has long been known that inducing additional random DSBs, for example by exposing cells to ionizing radiation, can increase the frequency of random integration (RI), however the irradiation doses that were used were non-physiological. We found that much smaller doses of radiation, in the upper range of radiological diagnostic procedures, stimulate integration even more efficiently than the much larger doses studied previously. The potency of stimulation is remarkable, given that biological effects of such low doses are generally difficult to register, and warrants re-evaluation using animal models. Surprisingly, the genetic dependencies of radiation-stimulated integration do not include DSB repair pathways, and are distinct from background integration events. Our observations provide a hyper-sensitive tool to detect mutagenesis and reveal new information about the genetic interactions between DNA damage signaling and repair system components. Show less
Naipal, K.A.T.; Verkaik, N.S.; Sanchez, H.; Deurzen, C.H.M. van; Bakker, M.A. den; Hoeijmakers, J.H.J.; ... ; Gent, D.C. van 2016
Artemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the... Show moreArtemis deficiency is known to result in classical T-B- severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical T-B- SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5' splice-site or an intronic point mutation creating a novel 3' splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 30 splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts. Genes and Immunity (2011) 12, 434-444; doi:10.1038/gene.2011.16; published online 10 March 2011 Show less
Wiegant, W.W.; Meyers, M.; Verkaik, N.S.; Burg, M. van der; Darroudi, F.; Romeijn, R.; ... ; Zdzienicka, M.Z. 2010
V(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through nonhomologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe... Show moreV(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through nonhomologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe combined immunodeficiency (RS-SCID). Here, a SCID patient, M3, is described witha-T-B+NK+ phenotype but without causative mutations in CD3 delta, epsilon, zeta or IL7R alpha, genes specifically involved in T cell development. Clonogenic survival of M3 fibroblasts showed an increased sensitivity to the DSB-inducing agents ionizing radiation and bleomycin, as well as the crosslinking compound, mitomycin C. We did not observe inactivating mutations in known NHEJ genes and results of various DSB-repair assays in G(1) M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G(2) phase of the cell cycle. Checkpoint analysis indicated functional G(1)/S and intra-S checkpoints after irradiation but impaired activation of the "early" G(2)/M checkpoint. Together these results indicate a novel class of RS-SCID patients characterized by the specific absence of T lymphocytes and associated with defects in G(2)-specific DSB repair. The pronounced G(2)/M radiosensitivity of the RS-SCID patient described here, suggests a defect in a putative novel and uncharacterized factor involved in cellular DNA damage responses and T cell development. (C) 2009 Elsevier B.V. All rights reserved. Show less
