Search

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.

G4 DNA motifs, which can form stable secondary structures called G-quadruplexes, are ubiquitous in eukaryotic genomes, and have been shown to cause genomic instability. Specialized helicases that unwind G-quadruplexes in vitro have been identified, and they have been shown to prevent genetic instability in vivo. In the absence of these helicases, G-quadruplexes can persist and cause replication fork stalling and collapse. Translesion synthesis (TLS) and homologous recombination (HR) have been proposed to play a role in the repair of this damage, but recently it was found in the nematode Caenorhabditis elegans that G4-induced genome alterations are generated by an error-prone repair mechanism that is dependent on the A-family polymerase Theta (Pol θ). Current data point towards a scenario where DNA replication blocked at G-quadruplexes causes DNA double strand breaks (DSBs), and where the choice of repair pathway that can act on these breaks dictates the nature of genomic alterations that are observed in various organisms.

Small RNA molecules participate in a variety of activities in the cell: in a process known as RNA interference (RNAi), double-stranded RNA triggers the degradation of messenger RNA that has a matching sequence; the small RNA intermediates of this process can also modify gene expression in the nucleus. Here we show that a single episode of RNAi in the nematode Caenorhabditis elegans can induce transcriptional silencing effects that are inherited indefinitely in the absence of the original trigger. Our findings may prove useful in the ongoing development of RNAi to treat disease.

Heterozygous germ-line mutations in BRCA1 and BRCA2 predispose to several types of cancer. Owing to their roles in the error-free repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), lack of BRCA1/2 in these tumors results in DNA damage defects that can be specifically targeted by the inhibition of Poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is a key sensor of DNA damage and its inhibition has been shown to be synthetically lethal with deficiencies in HR, resulting in the selective killing of BRCA1/2-deficient tumor cells, while sparing BRCA1/2-proficient non-tumor cells. The success of this approach has resulted in the approval of four PARP1 inhibitors (PARPi) for the treatment of ovarian, breast, prostate and pancreatic cancers. However, drug resistance poses a major obstacle as, despite initial responses, patients receiving PARPi often develop resistance to the treatment. Understanding the molecular mechanisms behind PARPi resistance is therefore crucial to identify key determinants of PARPi response and to find combination treatment strategies to overcome resistance to PARPi by preventing, delaying or targeting resistant clones. In this thesis, we expanded our insights into the molecular mechanisms underlying PARPi resistance by conducting functional genetic screens in PARPi-resistance cell lines.

Introduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis drive the conformational changes in MutS thatare needed for the mismatch repair cascade.Chapter 2 focuses on the final stages of the DNA mismatch repair pathway, which are the resection and subsequent resynthesis of the mismatch containing strand.Chapter 3 presents an example of how DNA polymerases can be targeted for the development of novel antibiotics against Mycobacterium tuberculosis (Mtb).Chapter 4 describes a new instrument named the Puffalot, developed for the preparation of cryo-EM grids, which aims to improve the reliability of cryo-EM sample preparation.Discussion provides a summary of the scientific findings described in this thesis in light of the published literature as well as an overview of the future directions and perspectives.

Introduction contains a general overview of the research topics discussed in this thesis.Chapter 1 addresses a fundamental question in DNA mismatch repair, which is how ATP binding and hydrolysis drive the conformational changes in MutS thatare needed for the mismatch repair cascade.Chapter 2 focuses on the final stages of the DNA mismatch repair pathway, which are the resection and subsequent resynthesis of the mismatch containing strand.Chapter 3 presents an example of how DNA polymerases can be targeted for the development of novel antibiotics against Mycobacterium tuberculosis (Mtb).Chapter 4 describes a new instrument named the Puffalot, developed for the preparation of cryo-EM grids, which aims to improve the reliability of cryo-EM sample preparation.Discussion provides a summary of the scientific findings described in this thesis in light of the published literature as well as an overview of the future directions and perspectives.

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 (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.

DNA encodes the genetic instructions for living organisms. However, damage to the DNA is inevitable, because DNA itself is an unstable molecule and environmental factors such as UV-radiation or X-rays cause damage to the DNA. A certain type of DNA damages can block DNA replication, an essential step before cell can divide. The polymerases that normally replicate DNA are incredibly efficient and virtually flawless on undamaged DNA, but they cannot replicate damaged DNA. In multi-celled organisms, the most important defense mechanism against this is Translesion DNA synthesis (TLS). TLS protects against various negative consequences of damage to the DNA. For this, TLS utilizes specialized TLS polymerases that can replicate damaged DNA.My experiments show that the strong evolutionary conservation of TLS is explained by the dual functions of TLS: guarding replication potential and genome stability. TLS suppresses genomic instability, by preventing conversion of replication blocks to double-stranded DNA breaks (DSBs). Without functional TLS, DSBs arise and result in larger and more harmful mutations. TLS is beneficial for organisms because it supports continuous reproduction and growth. Although DNA damage is always present and unavoidable, TLS guards against the formation of mutations that would otherwise lead to cancer, aging and congenital disease.

Inactivating mutations in BRCA1 or BRCA2 genes predispose to several types of cancer. Owing to their roles in maintaining genomic stability, lack of BRCA1/2 results in DNA damage repair defects, a vulnerability that can be exploited therapeutically by the inhibition of poly(ADPribose) polymerase 1 (PARP1). Unfortunately, clinical benefit of PARPi therapy is often limited by emerging drug resistance. Identification of PARPi resistance mechanisms is therefore crucial to improve the clinical outcome and design strategies that would ultimately prevent or target resistant tumors.The use of genetically engineered mouse models (GEMMs) of BRCA1/2-associated breastcancer in this work has allowed us to model PARPi resistance in vivo in well-defined genetic contexts. By combining high-throughput genetic screens, multiple omics analyses and functional assays, we identified several factors of PARPi resistance and explained their role in therapy failure. Moreover, we established a new tumor-derived organoid system thatenables robust in vivo validation of putative drug resistance factors. Finally, work described in this thesis has advanced our understanding of basic biological processes involved in DNA damage signaling and repair.

In forensic science, next to the question of who contributed to the trace (DNA analysis), knowledge regarding the cellular origin of the evidentiary trace, thus what cell type contributed to the trace, is often key to facilitate inference of activities. To this aid, RNA-based approaches have shown their up come in forensic investigations since 1999. RNA profiling techniques rely on the fact that different cell types, such as blood, express a characteristic pattern of genes, such as haemoglobin beta (HBB) expressed in the red blood cells. This thesis describes studies that assisted the expanding and advancement of forensic RNA profiling assays used for the inference of body fluids and organ tissues.

By investigating the interaction between different types of nucleases (i.e. nicking versus cleaving), donor DNA structures and target chromatin environments, this thesis provides important insights into how to improve the three crucial parameters of genome editing: efficiency, specificity and fidelity. The work presented in this thesis expand the range of possibilities for high-fidelity genetic manipulation of human cells.

The capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is therefore tightly controlled by regulatory mechanisms. Post-translational modifications (PTMs) are able to directly change the function of a protein and thereby provide a quick functional switch. This thesis focusses on the roles of small ubiquitin-like modifiers (SUMOs) and their crosstalk with other post-translational modifications during cell division, at the proteome-wide level as well as the single target protein level.

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, 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.

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.

Upon the induction of DNA damage, cells initiate a protective response, referred to as the DNA damage response (DDR), to repair DNA damage and maintain genome integrity. This response is driven and regulated by posttranslational protein modifications and chromatin remodeling events. Mutations or aberrant expression of chromatin modifying proteins not only impacts on the DDR, but also causes human diseases with severe clinical phenotypes, illustrating the importance of these proteins for genome stability maintenance and human health. Largely unclear is, however, which and how chromatin modifying enzymes control the complex DDR pathways and in this manner prevent the onset of disease. To this end, we employed cross-disciplinary approaches that combined cell biological, biochemical and microscopic methods to identify histone modifying enzymes, chromatin remodelers as well as other DDR proteins and elucidate their mechanistic role in the response to DNA doublestrand breaks (DSBs) and disease prevention.

Dit proefschrift beschrijft het onderzoek naar de stabiliteit van twee typen DNA volgordes (sequenties) die vaak voorkomen in DNA: microsatellieten en G-quadruplex sequenties. Microsatellieten zijn kleine stukjes repeterend DNA en G-quadruplex sequenties hebben de unieke eigenschap om een DNA-structuur te vormen die bestaat uit vier DNA-strengen. Bij een celdeling, waarbij het DNA gekopieerd moet worden, blijken deze twee sequenties soms lastig te kopi_ren te zijn. Dit kan tot DNA-instabiliteit leiden. Deze instabiliteit wordt in verband gebracht met kanker en neurodegeneratieve ziektes zoals ALS. Het is daarom van groot belang om alles te weten te komen over microsatelliet- en G-quadruplex-instabiliteit. Allereerst worden in dit proefschrift nieuwe methodes beschreven waarmee de instabiliteit van microsatellieten en G-quadruplexes makkelijk kan worden waargenomen. Met behulp van deze methodes zijn vervolgens verschillende ontdekkingen gedaan. Zo is bijvoorbeeld ontdekt dat in menselijke cellen een klein RNA-molecuul betrokken is bij het instabiel worden van microsatellieten en het ontstaan van darmkanker. Een ander belangrijke bevinding is de ontdekking van een nieuw soort DNA-reparatie mechanisme in de rondworm. De ontdekking van dit mechanisme, waarbij het eiwit polymerase theta G-quadruplex-ge_nduceerde DNA schade repareert, heeft tot nieuwe inzichten geleid op het gebied van genetische mutaties, evolutie en het bestrijden van tumoren.

The genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a DNA double-strand break (DSB) in which both strands of the helix are broken. When not repaired adequately DSBs can lead to extensive loss of genetic information and/or genomic rearrangements, ultimately fueling genome instability, cellular dysfunction and malignant transformation. This thesis describes several studies conducted to examine how living organisms preserve their genetic material and how different DNA repair pathways influence genome stability. To study these questions the nematode C. elegans was used as a model organism, as it allows efficient genetic manipulation as well as in-depth genetic analysis of mutagenic processes. We exploited these unique attributes to i) convert these animals into in vivo sensors of DNA damage ii) identify factors not implicated in genome stability before, iii) unveil mechanisms that dictate DNA repair pathway choice, and iv) determine the biological consequences of endogenous barriers that impede DNA replication.

In this thesis I describe the developmental role of the Y-family polymerases Pol Eta, Pol Kappa and Rev1 in protection against exogenous and endogenous damage in C. elegans. Furthermore I identify a new role for the A-family Polymerase Pol Theta in repair of replication-associated breaks.

DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little or no sequence homology, and this is often accompanied by the loss of a few nucleotides. Single-strand annealing (SSA) uses sequence homology within the same chromosome and leads to deletion of one of the repeats and the intervening sequence. Using the model organism C. elegans, we study DSB repair in the context of a developing animal and in complex genetic backgrounds. We make use of a transgenic approach where the restriction enzyme I-SceI can be expressed in an inducible manner, combined with a reporter transgene that contains the 18-nt recognition site for I-SceI in an out-of-frame LacZ gene. In Chapter 2 of this thesis, we use this assay to reveal the activity of a fourth pathway, which we termed alternative end-joining (alt-EJ). This pathway seems to act as a backup for NHEJ because it predominates repair only in the absence of canonical NHEJ, but its repair products are characterized by frequent use of homology in a way that is similar to SSA. Alt-EJ operates independently of many known repair genes and leads to very efficient DSB repair even in triple mutants that are defective for HR, SSA and NHEJ. Despite its putative function as a backup for classic NHEJ, we show in Chapter 3 that, in contrast to NHEJ, alt-EJ only occurs in replicating cells, leading to DSB persistence in non-replicating NHEJ-deficient somatic cells. Although normally highly toxic, endogenous DSBs are introduced in a regulated manner in meiotic cells in the germline. These DSBs need to be repaired by HR to establish crossover formation between homologous chromosomes which is required for genetic diversity among the offspring and for correct chromosome segregation. In Chapter 4 we show that besides HR, other repair pathways are also active in the germline. Moreover, the response to DSBs is highly dependent on the stage of the cell cycle at the time of DSB induction and differs between different germline zones. Quadruplex of G4 DNA is a stable secondary ssDNA structure that can form in particular G-rich sequences during DNA replication. In mutants for the gene dog-1 (mammalian FANCJ), spontaneous deletions arise at G4 DNA. These deletions always initiate immediately downstream of the G-rich sequence and end at various locations downstream. In Chapter 5, we show that these deletions are likely formed through DSB intermediates, because they resemble DSBs at other locations in many ways. Remarkably, these DSBs are not repaired by one of the canonical repair routes, but may instead be repaired by alt-EJ or another mechanism. In Chapter 6, we show how deletion formation in dog-1 mutant background can be used a tool to isolate deletion alleles of many C. elegans genes.

DNA is the carrier of genetic information. DNA is constantly damaged by, for example, UV light and X-rays. Cells can utilize a large number of proteins that can repair the damages, thereby avoiding changes in the DNA sequence. Damages that are not repaired result in an increase in the number of mutations. Mutations can lead to the transformation of a normal cell to a tumor cell. This becomes clear with the observation that genes involved in DNA repair are often mutated in tumors, and that genetic forms of cancer are related to mutations in these genes. In this thesis, several DNA repair mechanisms are studied. These studies are performed in the model organism C. elegans. The nematode C. elegans has many genes in common with humans and it can easily be used in large genetic studies. The studies have resulted in the identification and characterization of new genes involved in DNA repair.

Genome stability is closely linked to cancer. Most, if not all tumor cells show some form of genome instability, mutations can range from single point mutations to gross chromosomal rearrangements and aneuploidy. Genome instability is believed to be the driving force behind tumorigenesis. In order to discover genes that keep an organism's genome stable, we made use of the powerful genetics of the model organism C. elegans. In the nematode C. elegans, genes can systematically be silenced using RNA interference (RNAi). we setup several assays to monitor genome stability in C. elegans and developed a method to do high-throughput genome-wide screens. Several types of cancer show micro satellite instability, using genome-wide RNAi, we screened for genes that protect the genome against frame shift mutations, and indeed found othologs of known human cancer genes as well as novel genes. Next we screened for genes that protect against DNA double strand breaks, since improper processing of DNA breaks can result in gross chromosomal rearrangements. The method described in the paper allows the identification of synthetic lethal interactions in C. elegans using the combination of RNAi and a genetic mutant. Recently we did a screen for genes required for resistance against ionizing radiation. In this screen we found several orthologs of genes known to predispose to cancer, as well as genes not previously linked to cancer. We knocked down orthologous genes in a human cell line and indeed the human cells became radiation sensitive, proving functional conservation of the genes. The presence of known genome stability genes validates the experimental setup; novel genes are likely to play a role in tumor biology.