Marcel Tijsterman

Mutagenic Capacity of Endogenous G4 DNA Underlies Genome Instability in FANCJ-Defective C. elegans

To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2α

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFα protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFα) and aha-1 (HIFβ), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2α-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2α, but not HIF-1α. These results identify TWIST1 as a direct target gene of HIF-2α, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.

Genomes and G-Quadruplexes: For Better or for Worse

Genomic integrity is crucial for correct chromosome segregation and physiological rates of cell proliferation. Mutations, deletions and translocations, hallmarks of human tumors, drive the aberrant proliferation and metastatic behavior of cancer cells. These chromosomal rearrangements often occur at genomic sites susceptible to breakage during DNA replication, including regions with G-quadruplex (G4)-forming potential. G4s are stable secondary structures that guanine-rich single-stranded DNA can readily adopt in vitro. However, their formation in eukaryotic cells has remained elusive and thus a subject of debate ever since they were first described. Recent work has more convincingly implicated G4s in a variety of biological processes including telomere maintenance, gene expression, epigenetic regulation and DNA replication. However, the downside of employing thermodynamically very stable alternative DNA structures as regulatory entities lies in their potential to also interfere with normal DNA metabolic processes, such as transcription and replication, which require readability of each base to faithfully transmit genetic information. Indeed, it has become clear that G4 structures can pose prominent barriers to replication fork progression and that they are also intrinsically recombinogenic. Here, we discuss mechanisms that cells evolved to counteract these detrimental effects, thereby ensuring the faithful inheritance of G4-containing genomes.

FANCJ promotes DNA synthesis through G-quadruplex structures

Our genome contains many G‐rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G‐quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G‐quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G‐quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G‐quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G‐quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G‐quadruplexes.

DNA double-strand break repair in Caenorhabditis elegans

Faithful repair of DNA double-strand breaks (DSBs) is vital for animal development, as inappropriate repair can cause gross chromosomal alterations that result in cellular dysfunction, ultimately leading to cancer, or cell death. Correct processing of DSBs is not only essential for maintaining genomic integrity, but is also required in developmental programs, such as gametogenesis, in which DSBs are deliberately generated. Accordingly, DSB repair deficiencies are associated with various developmental disorders including cancer predisposition and infertility. To avoid this threat, cells are equipped with an elaborate and evolutionarily well-conserved network of DSB repair pathways. In recent years, Caenorhabditis elegans has become a successful model system in which to study DSB repair, leading to important insights in this process during animal development. This review will discuss the major contributions and recent progress in the C. elegans field to elucidate the complex networks involved in DSB repair, the impact of which extends well beyond the nematode phylum.

COM-1 Promotes Homologous Recombination during Caenorhabditis elegans Meiosis by Antagonizing Ku-Mediated Non-Homologous End Joining

Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1–deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.

Polymerase Θ is a key driver of genome evolution and of CRISPR/Cas9-mediated mutagenesis

Cells are protected from toxic DNA double-stranded breaks (DSBs) by a number of DNA repair mechanisms, including some that are intrinsically error prone, thus resulting in mutations. To what extent these mechanisms contribute to evolutionary diversification remains unknown. Here, we demonstrate that the A-family polymerase theta (POLQ) is a major driver of inheritable genomic alterations in Caenorhabditis elegans. Unlike somatic cells, which use non-homologous end joining (NHEJ) to repair DNA transposon-induced DSBs, germ cells use polymerase theta-mediated end joining, a conceptually simple repair mechanism requiring only one nucleotide as a template for repair. Also CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. Finally, through whole-genome sequencing of propagated populations, we show that only POLQ-proficient animals accumulate genomic scars that are abundantly present in genomes of wild C. elegans, pointing towards POLQ as a major driver of genome diversification.

A broad requirement for TLS polymerases η and κ, and interacting sumoylation and nuclear pore proteins, in lesion bypass during C. elegans embryogenesis.

Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS–induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS.

The repair of G-quadruplex-induced DNA damage.

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.