Preservation of lagging strand integrity at sites of stalled replication by pol α-primase and 9-1-1 complex

Plant science

Translesion synthesis polymerases are dispensable for C. elegans reproduction but suppress genome scarring by polymerase theta-mediated end joining

Author summaryResearch in the fields of DNA repair and mutagenesis has led to enormous insight into the mechanisms responsible for maintaining genetic integrity. However, which processes drive de novo mutations and will thus contribute to inherited diseases are still unclear. One process thought to underlie spontaneous mutagenesis is replication of damaged DNA by specialised so-called "Translesion synthesis" polymerases, which have the ability to replicate across damaged bases, but are not very accurate. To address the impact of TLS or the lack thereof on genome integrity, we have knocked out all TLS enzymes that are encoded by the C. elegans genome, individually and in combination, and monitored mutation accumulation during prolonged culturing of these animals without external sources of DNA damage. We found that TLS is not the major driver of spontaneous mutagenesis in this organism, however, it protects the genome from harmful small deletions that result from mutagenic repair of DNA breaks. We also found that, contrary to what was expected, TLS activity is not essential for reproduction in a multicellular organism with the tissue complexity and genome size of C. elegans.Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information.Genome Instability and Cance

Small tandem DNA duplications result from CST-guided Pol alpha-primase action at DNA break termini

Error-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA polymerase alpha primase plays part in tandem duplication formation at CRISPR/Cas9-induced complementary 3 ' ssDNA protrusions.Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase alpha (Pol alpha)-primase in tandem duplication formation at breaks having complementary 3 ' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3 ' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3 ' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.Genome Instability and Cance

Helicase Q promotes homology-driven DNA double-strand break repair and prevents tandem duplications

DNA double-strand breaks are a major threat to cellular survival and genetic integrity. In addition to high fidelity repair, three intrinsically mutagenic DNA break repair routes have been described, i.e. single-strand annealing (SSA), polymerase theta-mediated end-joining (TMEJ) and residual ill-defined microhomology-mediated end-joining (MMEJ) activity. Here, we identify C. elegans Helicase Q (HELQ-1) as being essential for MMEJ as well as for SSA. We also find HELQ-1 to be crucial for the synthesis-dependent strand annealing (SDSA) mode of homologous recombination (HR). Loss of HELQ-1 leads to increased genome instability: patchwork insertions arise at deletion junctions due to abortive rounds of polymerase theta activity, and tandem duplications spontaneously accumulate in genomes of helq-1 mutant animals as a result of TMEJ of abrogated HR intermediates. Our work thus implicates HELQ activity for all DSB repair modes guided by complementary base pairs and provides mechanistic insight into mutational signatures common in HR-defective cancers.Microhomology-mediated end-joining (MMEJ) is a poorly defined mutagenic DNA break repair pathway. Here the authors show that the helicase HELQ is essential for polymerase theta-independent MMEJ, single-strand annealing and homologous recombination through synthesis dependent strand annealing in C. elegans.Genome Instability and Cance

Gene targeting in polymerase theta-deficient Arabidopsis thaliana

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Pol theta)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Pol theta. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Pol theta in T-DNA integration. Pol theta deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.Genome Instability and Cance

Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches

Gene targeting in polymerase theta-deficient arabidopsis thaliana

Plant science

Silenced yeast chromatin is maintained by Sir2 in preference to permitting histone acetylations for efficient NER

Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER

Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses

Novel viruses have been discovered in wild Caenorahbditis nematode isolates and can now be used to explore host antiviral pathways, nematode ecology, and host-pathogen co-evolution

An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense