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DNA polymerase θ (POLQ) repairs mitotic DNA breaks; this requires RHINO and PLK1, averts genomic instability and may underlie effects of POLQ inhibitors in HDR-deficient cancer cells. We discuss recent work on mitotic DNA break processing and repair, the need for multiple DSB repair pathways and implications of therapeutic POLQ targeting in cancer.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant’s nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5′ end. Different from capture of the 3′ end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5′ attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5′ end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5′ end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3′ capture. Our study provides a mechanistic model for how Agrobacteriumexploits the plant’s own DNA repair machineries to transform it.

Organisms have developed specialized and intricate mechanisms to cope with environmental threats that depend on their natural habitat and ability to move. Arabidopsisdemonstrates an impressive adaptation using cryptochrome 1 to maintain genomic stability through a blue-light-mediated process that involves the recruitment of repair proteins to double-strand breaks.

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

Histone H3 lysine 9 (H3K9) methylation is a conserved modification that generally represses transcription. In Caenorhabditis elegans it is enriched on silent tissue-specific genes and repetitive elements. In met-2 set-25 double mutants, which lack all H3K9 methylation (H3K9me), embryos differentiate normally, although mutant adults are sterile owing to extensive DNA-damage-driven apoptosis in the germ line. Transposons and simple repeats are derepressed in both germline and somatic tissues. This unprogrammed transcription correlates with increased rates of repeat-specific insertions and deletions, copy number variation, R loops and enhanced sensitivity to replication stress. We propose that H3K9me2 or H3K9me3 stabilizes and protects repeat-rich genomes by suppressing transcription-induced replication stress.

Mosaic analysis, in which two or more populations of cells with differing genotypes are studied in a single animal, is a powerful approach to study developmental mechanisms and gene function in vivo. Over recent years, several genetic methods have been developed to achieve mosaicism in zebrafish, but despite their advances, limitations remain and different approaches and further refinements are warranted. Here, we describe an alternative approach for creating somatic mosaicism in zebrafish that relies on the instability of microsatellite sequences during replication. We placed the coding sequences of various marker proteins downstream of a microsatellite and out-of-frame; in vivo frameshifting into the proper reading frame results in expression of the protein in random individual cells that are surrounded by wild-type cells. We optimized this approach for the binary Gal4-UAS expression system by generating a driver line and effector lines that stochastically express Gal4-VP16 or UAS:H2A-EGFP and self-maintaining UAS:H2A-EGFP-Kaloop, respectively. To demonstrate the utility of this system, we stochastically expressed a constitutively active form of the human oncogene H-RAS and show the occurrence of hyperpigmentation and sporadic tumors within 5 days. Our data demonstrate that inducing somatic mosaicism through microsatellite instability can be a valuable approach for mosaic analysis and tumor induction in Danio rerio.

In Schizosaccharomyces pombe two different repair mechanisms remove UV-induced lesions from DNA, i.e. nucleotide excision repair (NER) and UV damage repair (UVDR). Here, the kinetics of removal of cyclobutane pyrimidine dimers (CPDs) by both pathways is determined at base resolution in the transcribed strand (TS) and the non-transcribed strand (NTS) of the sprpb2+ gene. UVDR does not remove lesions in a strand-specific manner, indicating that UVDR is neither stimulated nor inhibited by RNA polymerase II transcription. In contrast, in a UVDR-deficient strain the TS is repaired preferentially. This strong strand bias suggests that in S.pombe, as in other species, NER is coupled to transcription. In repair-proficient S.pombe the TS is repaired very rapidly, as a consequence of two efficiently operating pathways, while the NTS is repaired more slowly, mainly by UVDR. Furthermore, we demonstrate that UVDR is not always faster than NER.

The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

ABSTRACTThe nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA. NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions. Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens. In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3gene at nucleotide resolution. In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage. Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate. Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA. The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

The essential Saccharomyces cerevisiae KIN28gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair (NER). Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II complex. Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28allele. The removal of UV induced cyclobutane pyrimidine dimers (CPDs) was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2and URA3. Cells carrying the thermosensitive KIN28allele display a transcription-coupled repair (TCR) defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription. The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general.

Agrobacterium tumefaciensis a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown1. Here we demonstrate that in Arabidopsis thalianaT-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQmutant plants (which have mutated Pol θ), although susceptible to Agrobacteriuminfection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA–plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3′ end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer–template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

Repair of UV-induced cyclobutane pyrimidine dimers (CPDs) was examined at single nucleotide resolution in the yeast Saccharomyces cerevisiae, using an improved protocol for genomic end-labelling. To obtain the sensitivity required for adduct detection in yeast, an oligonucleotide-directed enrichment step was introduced into the current methodology developed for adduct detection in Escherichia coli. With this method, heterogeneous repair of CPDs within the RPB2 locus is observed. Individual CPDs positioned in the transcribed strand are removed very efficiently with identical kinetics. This fast repair starts within 23 bases downstream of the transcription initiation site. The non-transcribed strand of the active gene exhibits slow repair without detectable repair variations between individual lesions. In contrast, CPDs positioned in the promoter region show profound repair heterogeneity. Here, CPDs at specific sites are removed very quickly, with comparable rates to CPDs positioned in the transcribed strand, while at other positions lesions are not repaired at all during the period studied. Interestingly, the fast repair in the promoter region is dependent on the RAD7 and RAD16 genes, as are the slowly repaired CPDs in this region and in the non-transcribed strand. This indicates that the global genome repair pathway is not intrinsically slow and at specific positions can be as efficient as the transcription-coupled repair pathway.

The recently cloned Saccharomyces cerevisiae MMS19 gene appears to be involved in both nucleotide excision repair (NER) and transcription, which is also the case for components of the NER/transcription complex TFIIH. Unlike TFIIH however, the Mms19 protein does not affect NER in a highly purified in vitro system. In order to investigate the role of Mms19 in NER, we have analysed the repair capacity of the mms19 disruption mutant. We find that a cell-free extract of this mutant is deficient for NER in vitro. Since mms19 mutants are only moderately sensitive to irradiation with ultraviolet (UV) light, it is possible that such mutants are specifically deficient in one of the two modes of NER, i.e. transcription-coupled or global genome repair. To investigate this possibility, we have analysed the removal of cyclobutane-pyrimidine dimers (CPDs) at the nucleotide level in an mms19 mutant. Repair of CPDs was not detectable for both transcribed and non-transcribed sequences in this mutant, demonstrating a requirement for Mms19 in both transcription-coupled and global genome repair. Our data, combined with those obtained by others, suggest that Mms19 is required for NER in yeast, although it seems likely that the protein plays an indirect role in this process.