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
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
Caron, P.; Linden, J. van der; Attikum, H. van 2019
DNA double-strand breaks (DSBs) affect chromatin integrity and impact DNA-dependent processes such as transcription. Several studies revealed that the transcription of genes located in close... Show moreDNA double-strand breaks (DSBs) affect chromatin integrity and impact DNA-dependent processes such as transcription. Several studies revealed that the transcription of genes located in close proximity to DSBs is transiently repressed. This is achieved through the establishment of either a transient repressive chromatin context or eviction of the RNA polymerase II complex from the damaged chromatin. While these mechanisms of transcription repression have been shown to affect the efficiency and accuracy of DSB repair, it became evident that the transcriptional state of chromatin before DSB formation also influences this process. Moreover, transcription can be initiated from DSB ends, generating long non-coding (lnc)RNAs that will be processed into sequence-specific double-stranded RNAs. These so-called DNA damage-induced (dd)RNAs dictate DSB repair by regulating the accumulation of DNA repair proteins at DSBs. Thus, a complex interplay between mechanisms of transcription activation and repression occurs at DSBs and affects their repair. Here we review our current understanding of the mechanisms that coordinate transcription and DSB repair to prevent genome instability arising from DNA breaks in transcribed regions. Show less
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
Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA... Show moreAccurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequences flanked by DNA regions sharing sequence identity ("homologous") to those bracketing the target site. The strategies for mediating the formation of DSBs at the predefined genomic loci, have been undergoing intense investigation since the introduction of sequence-customizable zinc-finger nuclease (ZFN) technology. More recently, prokaryotic protein-based transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) derived from CRISPR-associated protein (Cas9) complexes have substantially broadened the availability and applicability of designer nuclease-mediated genome editing. A potential alternative research line to the use of designer nucleases, is to investigate whether specific DNA structures can, by themselves, serve as triggers of the DNA damage response and, in doing so, elicit targeted gene repair. Such an approach would simplify genome editing protocols, such as, by reducing the number of reagents needed to be introduced into target cells. In this thesis, the roles of these secondary structures as well as designer nucleases and donor-DNA templates, delivered via adenoviral vectors, is described. Show less
Agrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor... Show moreAgrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor-inducing (Ti) plasmid. As a result of the expression of Vir proteins, a section of this plasmid termed the T-DNA is transferred via a type four secretion system (T4SS) as a T-strand to the recipient cell where it can integrate into the genomic DNA of the recipient cell. In this thesis the prerequisites of T-DNA circle formation using yeast as a model were assayed. It was found that the homologous repair (HR) protein Rad52 is of importance to the formation of T-DNA circles. In addition to this, the effects of double strand break (DSB) induction on the efficiency of T-DNA integration via the HR pathway in yeast and the role the nucleosome occupancy of the target locus has on DSB induction were assayed. It was found that a decrease in the nucleosome occupancy of a target locus had no measurable impact on the efficiency of T-DNA integration at this locus. However, the combined translocation of nuclease proteins aimed at the target locus and T-strands during AMT greatly facilitated HR-mediated integration of T-DNA. Show less
All organisms are composed of cells and the cell's nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial... Show moreAll organisms are composed of cells and the cell's nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial importance. Double-strand breaks (DSBs) in DNA can be induced by ionising radiation and DNA damaging agents but also arise as intermediates in several cellular processes (e.g. meiosis). DSBs are among the most genotoxic DNA lesions and their accurate repair is crucial. Genetic instability resulting from unrepaired DSBs can lead to cell death and, in a multicellular organism, to cancer. There are two major DSB repair pathways: homologous recombination (HR) and non-homologous endjoining (NHEJ). By NHEJ, broken DNA is sealed together, irrespective of sequence homology, in a not necessarily error-free way. HR, in which a homologous DNA molecule is needed as a template, accurately repairs DSBs. In this thesis we mainly focus on recombinational repair proteins of the RAD52 epistasis group and involved in HR. We analyse the biochemical properties of two Rad52 homologs in S. pombe, Rad22A and Rad22B. We examine combined mutations in RAD52 and RAD54 homologs in S. pombe and mice. We investigate the importance of sumolyation of recombinational repair proteins. We also introduce mice deficient in Sycp1, important for coordination during meiosis and the formation of crossovers Show less
