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The Ku-binding motif (KBM) is a short peptide module first identified in APLF that we now show is also present in Werner syndrome protein (WRN) and in Modulator of retrovirus infection homologue (MRI). We also identify a related but functionally distinct motif in XLF, WRN, MRI and PAXX, which we denote the XLF-like motif. We show that WRN possesses two KBMs; one at the N terminus next to the exonuclease domain and one at the C terminus next to an XLF-like motif. We reveal that the WRN C-terminal KBM and XLF-like motif function cooperatively to bind Ku complexes and that the N-terminal KBM mediates Ku-dependent stimulation of WRN exonuclease activity. We also show that WRN accelerates DSB repair by a mechanism requiring both KBMs, demonstrating the importance of WRN interaction with Ku. These data define a conserved family of KBMs that function as molecular tethers to recruit and/or stimulate enzymes during NHEJ. Werner syndrome is a progeroid disease characterised by genetic instability due to mutations to the WRN helicase/exonuclease. Here the authors define a novel Ku binding motif (KBM) and show that two such motifs facilitate the involvement of WRN in DNA double-strand break repair.

Non‐homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double‐strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein–DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin‐and‐PNK‐Like Factor (APLF) into Ku‐DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4‐Lig4 and XLF, thereby assembling multi‐protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4‐Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway. In response to DNA double‐strand breaks, Aprataxin‐and‐PNK‐Like Factor (APLF) interacts with Ku80 and assembles a platform for sequential recruitment of several activities into vertebrate NHEJ repair complexes.

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break. Chromosomal single-strand DNA breaks occur frequently and require repair to avoid disease outcomes. Here, the authors show that in bird cells, PARP3 accelerates this repair, and use structural biology and cell biology techniques to reveal details of the mechanism of action.

Gene assembly of the variable domain of antigen receptors is initiated by DNA cleavage by the RAG1—RAG2 protein complex at sites flanking V, D, and J gene segments. Double-strand breaks are produced via a single-strand nick that is converted to a hairpin end on coding DNA and a blunt end on the neighboring recombination signal sequence. We demonstrate that the C-terminal regions of purified murine RAG1 (aa 1009—1040) and RAG2 (aa 388—520, including a plant homeodomain [PHD domain]) collaborate to inhibit the hairpinning stage of DNA cleavage. The C-terminal region of RAG2 stabilizes the RAG1/2 heterotetramer but destabilizes the RAG—DNA precleavage complex. This destabilization is reversed by binding of the PHD domain to a histone H3 peptide trimethylated on lysine 4 (H3K4me3). The addition of H3K4me3 likewise alleviates the RAG1/RAG2 C-terminus-mediated inhibition of hairpinning and the PHD-mediated inhibition of transposition activity. Thus a negative regulatory function of the noncore regions of RAG1/2 limits the RAG endonuclease activity in the absence of an activating methylated histone tail bound to the complex.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x‐ray radioresistance in HPV‐negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia‐inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF‐1α and HIF‐1β leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia‐induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

The rearrangement of antigen receptor genes is initiated by double-strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some \"cut-and-paste\" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation.

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.

ObjectiveTo evaluate a new protocol of risk stratification and early discharge for children with febrile neutropenia (FN).DesignProspective service evaluation from 17 April 2020 to 16 April 2021.Setting13 specialist centres in the UK.Patients405 children presenting with FN.InterventionAll children received intravenous antibiotics at presentation. Risk stratification was determined using the Australian-UK-Swiss (AUS) rule and eligibility for homecare assessed using criteria including disease, chemotherapy, presenting features and social factors. Those eligible for homecare could be discharged on oral antibiotics after a period of observation proportional to their risk group.Main outcome measuresMedian duration of admission and of intravenous antibiotics, and percentage of patients with positive blood cultures, significant infection, readmission within 7 days of initial presentation, intensive care unit (ICU) admission, death from infection and death from other causes.Results13 centres contributed 729 initial presentations of 405 patients. AUS rule scores were positively correlated with positive blood cultures, significant infection, ICU admission and death. 20% of children were eligible for homecare with oral antibiotics, of which 55% were low risk (AUS 0–1). 46% low-risk homecare eligible patients were discharged by 24 hours vs 2% homecare ineligible. Homecare readmission rates were 14% overall and 16% for low-risk cases (similar to a meta-analysis of previous studies). No child eligible for homecare was admitted to ICU or died.ConclusionsUse of the AUS rule and homecare criteria allow for safe early outpatient management of children with FN.