Mismatch repair (MMR) is a DNA repair system that corrects base-base misincorporations generated by the replicative DNA polymerases. Previous publications have shown the involvement of the MMR... Show moreMismatch repair (MMR) is a DNA repair system that corrects base-base misincorporations generated by the replicative DNA polymerases. Previous publications have shown the involvement of the MMR pathway in control of DNA damage responses. The work presented in this thesis suggests that the regulation of DNA damage induced mutagenesis and signaling may lie in the complex interplay between translesion synthesis (TLS), a subset of error-prone polymerases capable of replicating damaged DNA, and MMR. This thesis suggests that MMR may reduce DNA damage induced mutagenesis via two ways: (i) recruit the somewhat less error-prone TLS polymerases to the DNA lesion and (ii) post-replicative removal of mismatched nucleotides. Of note, inheriting a heterogenous defect in an MMR gene results in increased risk of developing cancer, in particular colorectal cancer. The reason for this specific cancer tropism is, as of yet, unclear. The colorectal tract is continuously exposed to DNA damaging agents and indeed data in this thesis shows that MMR is important in protecting against diet-derived DNA damaging agents by controlling DNA damage induced mutagenesis and signaling. As such the colorectal cancer tropism of MMR of LS may partly be explained by the role of MMR in controlling the DNA damage response. Show less
Pathogenic variants in PALB2 and CHEK2 are associated with an increased risk of breast cancer. By contrast, for missense variants of uncertain significance (VUS) in these genes, the associated... Show morePathogenic variants in PALB2 and CHEK2 are associated with an increased risk of breast cancer. By contrast, for missense variants of uncertain significance (VUS) in these genes, the associated breast cancer risk is often unclear. To aid in their clinical classification, functional assays that determine the impact of missense VUS on PALB2 and CHK2 protein function have been performed in this thesis. By means of these functional analyses, numerous missense VUS (in both PALB2 and CHEK2) have been identified that are, from a functional viewpoint, just as damaging as known pathogenic (i.e., truncating) variants. In agreement, we observe that the level of impaired protein function correlates with the degree of increased breast cancer risk. Overall, these findings suggest that damaging PALB2 and CHEK2 missense VUS are associated with a risk of breast cancer similar to that of protein-truncating variants in these genes. This indicates the urgency of expanding the functional characterization of missense VUS in both PALB2 and CHEK2 to further understand the associated cancer risk. Show less
Heuvel, D. van den; Kim, M.; Wondergem, A.P.; Meer, P.J. van der; Witkamp, M.; Lambregtse, F.; ... ; Luijsterburg, M.S. 2023
XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways.... Show moreXPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum ( XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPAH244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (similar to 50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER. Show less
This thesis addresses the repair of DNA double-strand breaks (DSBs) that arise in different contexts, both artificially inflicted DNA damage and spontaneously arising breaks. We have found that the... Show moreThis thesis addresses the repair of DNA double-strand breaks (DSBs) that arise in different contexts, both artificially inflicted DNA damage and spontaneously arising breaks. We have found that the (mutational) repair outcome of a DSB depends on the context in which it occurs. When cells are not replicating, DSBs are repaired via non-homologous end-joining (NHEJ). NHEJ efficiency can be affected by defective RNA processing. In replicating cells, the preferable mechanism for DSB repair is homologous recombination (HR). When canonical HR cannot be executed, because the repair template is not available (at G4-induced breaks, for example) or when not all HR factors are present (in BRCA1 deficient situations), alternative annealing is needed. This is carried out via polymerase theta-mediated end-joining (TMEJ), or when homologous nucleotides are available, via HELQ-1 mediated annealing of these homologous stretches. Finally, we have found that large tandem duplications can arise when break ends cannot anneal properly after the extension step in HR. Show less
This thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in... Show moreThis thesis aims to gain a better understanding of NER, to elucidate new molecular mechanisms and proteins that orchestrate how DNA repair is carried out on genomic DNA that is tightly packed in chromatin inside the living cell. It is important to obtain a better clinical picture of how inherited defects in DNA repair genes shapes phenotypes in patients with DNA repair-deficiency disorders. Show less
DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI... Show moreDNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase zeta (Pol zeta)-mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error-free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error-free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Pol zeta and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR-mediated ICL repair is defective, and breaks are instead re-ligated by polymerase theta-dependent microhomology-mediated end-joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error-free ICL repair. Show less
Bloom Syndrome (BS) is a genetic DNA repair disorder, caused by mutations in the BLM gene. The clinical phenotype includes growth retardation, immunodeficiency and a strong predisposition to... Show moreBloom Syndrome (BS) is a genetic DNA repair disorder, caused by mutations in the BLM gene. The clinical phenotype includes growth retardation, immunodeficiency and a strong predisposition to different types of malignancies. Treatment of malignancies in BS patients with radiotherapy or chemotherapy is believed to be associated with increased toxicity, but clinical and laboratory data are lacking. We collected clinical data of two Dutch BS patients with solid tumors. Both were treated with radiotherapy before the diagnosis BS was made and tolerated this treatment well. In addition, we collected fibroblasts from BS patients to perform in vitro clonogenic survival assays to determine radiosensitivity. BS fibroblasts showed less radiosensitivity than the severely radiosensitive Artemis fibroblasts. Moreover, studies of double strand break kinetics by counting 53BP1 foci after irradiation showed similar patterns compared to healthy controls. In combination, the clinical cases and laboratory experiments are valuable information in the discussion whether radiotherapy is absolutely contraindicated in BS, which is the Case in other DNA repair syndromes like Ataxia Telangiectasia and Artemis. Show less
Garrelfs, M.R.; Takada, S.; Kamsteeg, E.J.; Pegge, S.; Mancini, G.; Engelen, M.; ... ; Willemsen, M.A. 2020
Background: We aimed to expand the number of currently known pathogenic PNKP mutations, to study the phenotypic spectrum, including radiological characteristics and genotype-phenotype correlations,... Show moreBackground: We aimed to expand the number of currently known pathogenic PNKP mutations, to study the phenotypic spectrum, including radiological characteristics and genotype-phenotype correlations, and to assess whether immunodeficiency and increased cancer risk are part of the DNA repair disorder caused by mutations in the PNKP gene.Methods: We evaluated nine patients with PNKP mutations. A neurological history and examination was obtained. All patients had undergone neuroimaging and genetic testing as part of the prior diagnostic process. Laboratory measurements included potential biomarkers, and, in the context of a DNA repair disorder, we performed a detailed immunologic evaluation, including B cell repertoire analysis.Results: We identified three new mutations in the PNKP gene and confirm the phenotypic spectrum of PNKP-associated disease, ranging from microcephaly, seizures, and developmental delay to ataxia with oculomotor apraxia type 4. Irrespective of the phenotype, alpha-fetoprotein is a biochemical marker and increases with age and progression of the disease. On neuroimaging, (progressive) cerebellar atrophy was a universal feature. No clinical signs of immunodeficiency were present, and immunologic assessment was unremarkable. One patient developed cancer, but this was attributed to a concurrent von Hippel-Lindau mutation.Conclusions: Immunodeficiency and cancer predisposition do not appear to be part of PNKP-associated disease, contrasting many other DNA repair disorders. Furthermore, our study illustrates that the previously described syndromes microcephaly, seizures, and developmental delay, and ataxia with oculomotor apraxia type 4, represent the extremes of an overlapping spectrum of disease. Cerebellar atrophy and elevated serum alpha-fetoprotein levels are early diagnostic findings across the entire phenotypical spectrum. (C) 2020 The Author(s). Published by Elsevier Inc. Show less
Gorte, J.; Beyreuther, E.; Danen, E.H.J.; Cordes, N. 2020
Pancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to... Show morePancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to surgery and chemotherapy. Owing to radiosensitive abdominal organs, high-precision proton beam radiotherapy has been regarded as superior to photon radiotherapy. To further elucidate the potential of combination therapies, we employed a more physiological 3D, matrix-based cell culture model to assess tumoroid formation capacity after photon and proton irradiation. Additionally, we investigated proton- and photon-irradiation-induced phosphoproteomic changes for identifying clinically exploitable targets. Here, we show that proton irradiation elicits a higher efficacy to reduce 3D PDAC tumoroid formation and a greater extent of phosphoproteome alterations compared with photon irradiation. The targeting of proteins identified in the phosphoproteome that were uniquely altered by protons or photons failed to cause radiation-type-specific radiosensitization. Targeting DNA repair proteins associated with non-homologous endjoining, however, revealed a strong radiosensitizing potential independent of the radiation type. In conclusion, our findings suggest proton irradiation to be potentially more effective in PDAC than photons without additional efficacy when combined with DNA repair inhibitors. Show less
Background The introduction of cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) with either oxaliplatin or mitomycin C for patients with colorectal... Show moreBackground The introduction of cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) with either oxaliplatin or mitomycin C for patients with colorectal peritoneal metastasis (CPM) has resulted in a major increase in overall survival. Nonetheless, despite critical patient selection, the majority of patients will develop recurrent disease within one year following CRS + HIPEC. Therefore, improvement of patient and treatment selection is needed and may be achieved by the incorporation of genetic biomarkers. This systematic review aims to provide an overview of genetic biomarkers in the DNA repair pathway that are potentially predictive for treatment outcome of patients with colorectal peritoneal metastases treated with CRS + HIPEC with oxaliplatin or mitomycin C. Methods A systematic review was conducted according to the PRISMA guidelines. Given the limited number of genetic association studies of intraperitoneal mitomycin C and oxaliplatin in patients with CPM, we expanded the review and extrapolated the data from biomarker studies conducted in colorectal cancer patients treated with systemic mitomycin C- and oxaliplatin-based chemotherapy. Results In total, 43 papers were included in this review. No study reported potential pharmacogenomic biomarkers in patients with colorectal cancer undergoing mitomycin C-based chemotherapy. For oxaliplatin-based chemotherapy, a total of 26 genetic biomarkers within 14 genes were identified that were signi?cantly associated with treatment outcome. The most promising genetic biomarkers wereERCC1rs11615,XPCrs1043953,XPDrs13181,XPGrs17655,MNATrs3783819/rs973063/rs4151330, MMR status, ATM protein expression,HIC1tandem repeat D17S5, andPIN1rs2233678. Conclusion Several genetic biomarkers have proven predictive value for the treatment outcome of systemically administered oxaliplatin. By extrapolation, these genetic biomarkers may also be predictive for the efficacy of intraperitoneal oxaliplatin. This should be the subject of further investigation. Show less
Boonen, R.A.C.M.; Vreeswijk, M.P.G.; Attikum, H. van 2020
In recent years it has become clear that pathogenic variants inPALB2are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of... Show moreIn recent years it has become clear that pathogenic variants inPALB2are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) inPALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS inPALB2. A combination of assays has been used to assess the impact ofPALB2VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis ofPALB2VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS inPALB2, as well as the functional characterization of variants that affectPALB2RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor- or platinum-based chemotherapy. Show less
Ciosi, M.; Maxwell, A.; Cumming, S.A.; Moss, D.J.H.; Alshammari, A.M.; Flower, M.D.; ... ; Enroll-HD Team 2019
Increasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases... Show moreIncreasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases such as CRISPR/Cas9 to induce DNA double strand breaks at target loci and activate repair via homologous recombination (HR). GT can then be achieved by HR-mediated integration of an artificial repair template, sharing homology with the target locus. Further improvements have been made with the in planta GT method, in which the repair template is pre-inserted in the genome and can be excised by nucleases. Although these improvements led to substantial increases in GT efficiency, GT is still not efficient enough to be feasible for crop biotechnology. This thesis describes strategies to further improve GT efficiency in the model plant Arabidopsis thaliana. One of these strategies was to perform in planta GT in meiocytes, cells that already have a higher rate of HR. Another strategy was to find new Arabidopsis mutants with increased GT frequencies and to identify genes involved in this phenotype. In the end, this may lead to a better understanding of the mechanisms underlying GT and these may be used to realize higher GT frequencies in plants. Show less
Double-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence,... Show moreDouble-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence, trigger various downstream events and finally bring about repair. Two main pathways are used for DNA DSB repair: Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ). Both of them function together to maintain genome integrity. At least two NHEJ pathways have been identified: the classic NHEJ pathway (c-NHEJ) and the backup-NHEJ pathway (b-NHEJ) also called alternative-NHEJ (a-NHEJ) or microhomology-mediated end-joining (MMEJ). Agrobacterium tumefaciens is widely used as a vector to produce genetically modified plants. Agrobacterium-mediated genetic transformation involves the transfer of T-DNA from its tumor-inducing plasmid to the host cell nucleus, where it integrates into the plant genome. However, the molecular mechanism of T-DNA integration is still unclear. T-DNAs can integrate at artificially induced DSBs, which suggests that DSB repair mechanisms are probably involved in T-DNA integration in plants. Arabidopsis NHEJ mutants have subsequently been studied for T-DNA integration. However, the results obtained by different research groups were variable and revealed either no or limited negative effects. Show less
Swuec, P.; Renault, L.L.R.; Borg, A.; Shah, F.; Murphy, V.J.; Twest, S. van; ... ; Costa, A. 2017
Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core... Show moreActivation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs. Show less
DNA is arguably the most important molecule found in any organism, as it contains all information to perform cellular functions and enables continuity of species. It is continuously exposed... Show more DNA is arguably the most important molecule found in any organism, as it contains all information to perform cellular functions and enables continuity of species. It is continuously exposed to DNA-damaging agents both from endogenous and exogenous sources. To protect DNA against these sources of DNA damage various DNA-repair mechanisms have evolved. If not properly repaired, DNA damage can lead to mutations that may eventually lead to cell-death or tumorigenesis. One of the most dangerous types of DNA damage is a DNA double-stranded break (DSB), in which a DNA molecule is broken into two pieces. Cells are equipped with several DSB-repair mechanisms to deal with this type of damage. Some of these mechanisms repair DSBs in an error-free fashion, while others are error-prone and can lead to the accumulation of mutations. Although accumulating many mutations in cells can lead to severely reduced cellular fitness, perfect DNA repair is less desirable in the long term as mutations allow for speciation and evolution to take place. The key question addressed in my thesis is which DSB-repair mechanisms organisms use to protect their genome against DSBs and I find alternative end-joining of DNA breaks to play a major role in maintaining genome stability. Show less
