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

Dupilumab provides sustained effectiveness on patient-reported outcomes and favorable safety in patients with moderate-to-severe atopic dermatitis: up to 5-year results from the daily practice BioDay Registry

Background: Long-term daily practice data on patient-reported benefits of dupilumab for atopic dermatitis (AD) remains limited. Objective: To evaluate patient-reported outcome measures (PROMs) and the safety of dupilumab in patients with moderate-to-severe AD over a follow-up period of up to 5 years. Methods: Data were extracted from the prospective, multicenter BioDay registry (October 2017–2022) of patients with moderate-to-severe AD treated with dupilumab in daily practice. Results: In total 1223 patients, 1108 adults and 115 pediatric patients were included. After ≥1 year of treatment, mean Patient-Oriented Eczema Measure (POEM), Dermatology Life Quality Index (DLQI), Numeric rating scale (NRS)-pruritus ranged between 7.8 and 8.7, 3.5 and 4.2, and 2.9 and 3.1 in adults, respectively, whilst these patient-reported outcome measures (PROMs) ranged between 8.9 and 10.9, 4.4 and 6.4, and 3.0 and 3.7 in pediatric patients, respectively. At follow-up, overall work impairment decreased from 40.1% to 16.3% to 13.3% in adults. Furthermore, class I obesity and itch-dominant patients generally had less favorable treatment response. Of all patients, 66.8% reported ≥1 adverse event, with conjunctivitis being the most common (33.7%). Limitations: The overall percentage of missing values for selected PROMs was 26% in adults and 46% in pediatric patients. Conclusion: In addition to favorable safety, dupilumab has demonstrated sustained effectiveness across various PROMs, underscoring the treatment benefits from patients' perspectives

Reporter system for homologous recombination in <i>C. elegans</i>.

<p>(A) Schematic representation of the reporter transgenes. Expression of the yeast endonuclease ISceI fused to Cherry is controlled by the heat shock promotor (pHS). The reporter transgene is placed behind the intestinal <i>elt-2</i> promotor (pElt-2). Upon activation of ISceI following heat shock, the ISceI endonuclease cuts into the GFP coding sequence. Repair by gene conversion from an aborted copy of GFP results again in full length GFP. (B) Expression of GFP and Cherry in worms containing the reporter transgenes. Upon heat shock induction all intestinal cells express cherry::ISceI. Repair of the break site by HR from an aborted GFP template results in GFP expression in some intestinal cells. (C) Quantification of the fraction of GFP positive worms in different genetic backgrounds. Each bar represents the mean of three independent experiments. Error bars denote the s.e.m. (D) Germline sensitivity of <i>polh-1 (lf31)</i>, <i>xpa-1(ok698)</i> and double mutants to γ-irradiation. Each line represents the mean of minimal three independent experiments. The percentage of surviving progeny is relative to the fraction of surviving progeny without any irradiation, since <i>polh-1(lf31)</i> and <i>xpa-1(ok698)</i> show about 30% synthetic lethality. Error bars denote s.e.m.</p

Lig4 and Rad54 Are Required for Repair of DNA Double-Strand Breaks Induced by P-Element Excision in Drosophila

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2–8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4

Germline sensitivity of <i>polh-1</i> and <i>polk-1</i> mutants to different sources of DNA damage.

<p>(A) Sensitivity to UV-irradiation. (B) Sensitivity to γ-irradiation. (C) Sensitivity to cisplatin. Adults were exposed to DNA damaging treatments and survival was quantified by counting dead embryos versus living progeny in the next generation. Each line represents the mean of minimal three independent experiments. Error bars denote the s.e.m.</p

Y-family polymerases POLH-1 and POLK-1 of <i>C. elegans</i>.

<p>(A) Phylogenetic tree displaying Y-family polymerases from <i>C. elegans, S. cerevisiae, D. melanogaster, E. coli</i> and <i>H. sapiens</i>. Respectively red and blue branches show <i>C. elegans</i> POLH-1 (Polη) and POLK-1 (Polκ). (B) Gene structure of the <i>C. elegans polh-1</i> and <i>polk-1</i> genes and the molecular nature of the alleles used in this study.</p

<i>polh-1</i> and <i>polk-1</i> protect in a redundant fashion against the methylating agent MMS.

<p>(A) Double mutants of <i>polh-1</i> and <i>polk-1</i> are severely sensitized to MMS exposure. Results of a representative experiment are shown. Error bars denote SD. (B) Delayed progression through the first embryonic division after MMS exposure in <i>polk-1</i> mutants. The interval between passing of the paternal pronucleus over the midline till the start of cytokinesis is timed for at least 5 embryos per datapoint. Statistical significance for the difference in delay between N2 and <i>polk-1</i> embryos treated with MMS was calculated with a student's t-test (p = 0.012). (C–J) RAD51 immunostainings of early embryos treated with MMS. Morphology of <i>polh-1; polk-1</i> double mutant embryos is abnormal after MMS exposure, displaying chromatin bridges and abundant RAD51 staining (J). <i>polh-1(ok3317)</i> and <i>polk-1(lf29)</i> single mutants show incidental RAD51 foci in embryos (F and H), while such foci were never observed in untreated controls (C, E, G and I).</p

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1

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