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The attentional blink reflects a ubiquitous bottleneck with selecting and processing the second of two targets that occur in close temporal proximity. An extensive literature has examined the attention blink as a unitary phenomenon. As a result, which specific component of attention – perceptual sensitivity, choice bias, or both – is compromised during the attentional blink, and their respective neural bases, remains unknown. Here, we address this question with a multialternative task and novel signal detection model, which decouples sensitivity from bias effects. We find that the attentional blink impairs specifically one component of attention – sensitivity – while leaving the other component – bias – unaffected. Distinct neural markers of the attentional blink were mapped onto distinct subcomponents of the sensitivity deficits. Parieto-occipital N2p and P3 potential amplitudes characterized target detection deficits, whereas long-range high-beta band (20–30 Hz) coherence between frontoparietal electrodes signaled target discrimination deficits. We synthesized these results with representational geometry analysis. The analysis revealed that detection and discrimination deficits were encoded along separable neural dimensions, whose configural distances robustly correlated with the neural markers of each. Overall, these findings provide detailed insights into the subcomponents of the attentional blink and reveal dissociable neural bases underlying its detection and discrimination bottlenecks.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing. Cas9 remains bound to DNA after cleavage and its removal is required for DNA double-strand break repair. Here, the authors show that the HLTF translocase disrupts the Cas9- DNA post-cleavage complexes in a process that requires the HLTF HIRAN domain and ATPase activity.

Kristijan Ramadan and colleagues report the identification of three individuals from two families with biallelic inactivating mutations in SPRTN causing early onset hepatocellular carcinoma and defects in the DNA replication stress response. Functional studies confirmed critical roles for SPRTN in G2/M checkpoint response and DNA replication. Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1 ) 1 , 2 , 3 , 4 , 5 , 6 , 7 in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis 1 , 2 , 3 , 4 , 5 , 6 , 7 . Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

The SPRTN metalloprotease is essential for DNA-protein crosslink (DPC) repair and DNA replication in vertebrate cells. Cells deficient in SPRTN protease exhibit DPC-induced replication stress and genome instability, manifesting as premature ageing and liver cancer. Here, we provide a body of evidence suggesting that SPRTN activates the ATR-CHK1 phosphorylation signalling cascade during physiological DNA replication by proteolysis-dependent eviction of CHK1 from replicative chromatin. During this process, SPRTN proteolyses the C-terminal/inhibitory part of CHK1, liberating N-terminal CHK1 kinase active fragments. Simultaneously, CHK1 full length and its N-terminal fragments phosphorylate SPRTN at the C-terminal regulatory domain, which stimulates SPRTN recruitment to chromatin to promote unperturbed DNA replication fork progression and DPC repair. Our data suggest that a SPRTN-CHK1 cross-activation loop plays a part in DNA replication and protection from DNA replication stress. Finally, our results with purified components of this pathway further support the proposed model of a SPRTN-CHK1 cross-activation loop. Cells deficient in SPRTN protease activity exhibit severe DNA-protein crosslink induced replication stress and genome instability. Here the author reveal a functional link between the SPRTN protease and the CHK1 kinase during physiological DNA replication.

The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. Cdc48/p97 is a key component of the ubiquitin-proteasome system, acting as a ubiquitin-directed segregase to regulate multiple cellular functions. Here the authors identify UBXN-3/FAF1 as a crucial regulator of chromatin-associated protein degradation that recruits Cdc48/p97 to DNA replication forks.

Glycosylation has been recognized as one of the most prevalent and complex post-translational modifications of proteins involving numerous enzymes and substrates. Its effect on the protein conformational transitions is not clearly understood yet. In this study, we have examined the effect of glycosylation on protein stability using molecular dynamics simulation of legume lectin soybean agglutinin (SBA). Its glycosylated moiety consists of high mannose type N-linked glycan (Man 9 GlcNAc 2 ). To unveil the structural perturbations during thermal unfolding of these two forms, we have studied and compared them to the experimental results. From the perspective of dynamics, our simulations revealed that the nonglycosylated monomeric form is less stable than corresponding glycosylated form at normal and elevated temperatures. Moreover, at elevated temperature thermal destabilization is more prominent in solvent exposed loops, turns and ends of distinct β sheets. SBA maintains it folded structure due to some important saltbridges, hydrogen bonds and hydrophobic interactions within the protein. The reducing terminal GlcNAc residues interact with the protein residues VAL161, PRO182 and SER225 via hydrophobic and via hydrogen bonding with ASN 9 and ASN 75. Our simulations also revealed that single glycosylation (ASN75) has no significant effect on corresponding cis peptide angle orientation. This atomistic description might have important implications for understanding the functionality and stability of Soybean agglutinin.

PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα) and systemic (circulating chemokines and cytokines) inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

Nature Communications 7: Article number: 10612 (2016); Published: 4 February 2016; Updated: 17 May 2016. This Article contains errors in the labelling of Fig. 5 and Supplementary Fig. 6, the former of which was introduced during the production process. In Fig. 5b, an ‘x’ indicating the presence of UBXN-3(SGG)-HIS in lane 6 was inadvertently omitted.

The attentional blink reflects a ubiquitous bottleneck with selecting and processing the second of two targets that occur in close temporal proximity. An extensive literature has examined the attention blink as a unitary phenomenon. As a result, which specific component of attention – perceptual sensitivity, choice bias, or both – is compromised during the attentional blink, and their respective neural bases, remains unknown. Here, we address this question with a multialternative task and novel signal detection model, which decouples sensitivity from bias effects. We find that the attentional blink impairs specifically one component of attention – sensitivity – while leaving the other component – bias – unaffected. Distinct neural markers of the attentional blink were mapped onto distinct subcomponents of the sensitivity deficits. Parieto-occipital N2p and P3 potential amplitudes characterized target detection deficits, whereas long-range high-beta band (20–30 Hz) coherence between frontoparietal electrodes signaled target discrimination deficits. We synthesized these results with representational geometry analysis. The analysis revealed that detection and discrimination deficits were encoded along separable neural dimensions, whose configural distances robustly correlated with the neural markers of each. Overall, these findings provide detailed insights into the subcomponents of the attentional blink and reveal dissociable neural bases underlying its detection and discrimination bottlenecks.

DNA double-strand break (DSB) repair by homologous recombination is initiated by DNA end resection, a process involving the controlled degradation of the 5′-terminated strands at DSB sites 1 , 2 . The breast cancer suppressor BRCA1–BARD1 not only promotes resection and homologous recombination, but it also protects DNA upon replication stress 1 , 3 , 4 , 5 , 6 , 7 , 8 – 9 . BRCA1–BARD1 counteracts the anti-resection and pro-non-homologous end-joining factor 53BP1, but whether it functions in resection directly has been unclear 10 , 11 , 12 , 13 , 14 , 15 – 16 . Using purified recombinant proteins, we show here that BRCA1–BARD1 directly promotes long-range DNA end resection pathways catalysed by the EXO1 or DNA2 nucleases. In the DNA2-dependent pathway, BRCA1–BARD1 stimulates DNA unwinding by the Werner or Bloom helicase. Together with MRE11–RAD50–NBS1 and phosphorylated CtIP, BRCA1–BARD1 forms the BRCA1–C complex 17 , 18 , which stimulates resection synergistically to an even greater extent. A mutation in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT repeats of BRCA1 and hence the integrity of the BRCA1–C complex 19 , 20 – 21 , inhibits resection, showing that BRCA1–C is a functionally integrated ensemble. Whereas BRCA1–BARD1 stimulates resection in DSB repair, it paradoxically also protects replication forks from unscheduled degradation upon stress, which involves a homologous recombination-independent function of the recombinase RAD51 (refs. 4 , 5 – 6 , 8 ). We show that in the presence of RAD51, BRCA1–BARD1 instead inhibits DNA degradation. On the basis of our data, the presence and local concentration of RAD51 might determine the balance between the pronuclease and the DNA protection functions of BRCA1–BARD1 in various physiological contexts. BRCA1–BARD1 directly promotes double-strand break repair by stimulating long-range DNA end resection pathways.