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
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... Show moreDNA 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. 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
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... Show moreDit 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. Show less
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... Show moreThe 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. Show less
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... Show moreIn 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. Show less
