Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells.

Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance.

Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway.

Etoposide (ETO) is an anticancer drug that targets topoisomerase II (TOP2). It stabilizes a normally transient TOP2-DNA covalent complex (TOP2cc), thus leading to DNA double-strand breaks (DSBs). Tyrosyl-DNA phosphodiesterases two (TDP2) is directly involved in the repair of TOP2cc by removing phosphotyrosyl peptides from 5'-termini of DSBs. Recent studies suggest that additional factors are required for TOP2cc repair, which include the proteasome and the zinc finger protein associated with TDP2 and TOP2, named ZATT. ZATT may alter the conformation of TOP2cc in a way that renders the accessibility of TDP2 for TOP2cc removal. In this study, our genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens revealed that ZATT also has a TDP2-independent role in promoting cell survival following ETO treatment. ZATT KO cells showed relatively higher ETO sensitivity than TDP2-KO cells, and ZATT/TDP2 DKO cells displayed additive hypersensitivity to ETO treatment. The study using a series of deletion mutants of ZATT determined that the N-terminal 1-168 residues of ZATT are required for interaction with TOP2 and this interaction is critical to ETO sensitivity. Moreover, depletion of ZATT resulted in accelerated TOP2 degradation after ETO or cycloheximide (CHX) treatment, suggesting that ZATT may increase TOP2 stability and likely participate in TOP2 turnover. Taken together, this study suggests that ZATT is a critical determinant that dictates responses to ETO treatment and targeting. ZATT is a promising strategy to increase ETO efficacy for cancer therapy.

Proteome-wide Analysis Reveals Substrates of E3 Ligase RNF146 Targeted for Degradation.

Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the ß-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.

Molecular determinants of polyubiquitin recognition by continuous ubiquitin-binding domains of Rad18.

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub2-Ub4) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the "extended LR motif" (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub2 model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub2, not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub2 more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.

Chemesthesis from volatile organic compounds: Psychophysical and neural responses.

In Experiment 1, subjects sought to localize the nostril stimulated, left or right, in tests with nine esters (acetates, propionates, and butyrates) at concentrations meant to trigger chemesthesis (pungency, irritation). The task produced psychometric functions for chemesthetic detection unconfounded by olfactory sensations. The functions indicated a sharp transition from no detection to perfect detection, rather uniform across the esters, which themselves varied in potency by two log units. The correlation between the thresholds for the eight materials that yielded thresholds and predictions from a published linear free energy relationship (LFER) equaled 0.99. In Experiment 2, amplitude of the negative mucosal potential (NMP) was recorded from the septum. The resulting functions also increased with concentration sharply. Against a criterion amplitude of the NMP, thresholds measured in the first experiment (and predictions from the LFER) correlated 0.99. The NMP seems to offer an adequate objective measure of sensory irritation. The LFER, although effective predictively, could stand to have a parameter to anticipate that molecules beyond a certain size fail to trigger irritation. In the present case, a cut-off of chemesthetic potency occurred between butyl butyrate and hexyl butyrate for the group of subjects, with some variation of the boundary among individuals.

Adsorption and isotope effects by ion exchange with 1-aza-12-crown-4 bonded Merrifield peptide resin.

Magnesium isotope effects were investigated by chemical ion exchange with synthesized 1-aza-12-crown-4 bonded Merrifield peptide resin using elution chromatography. The capacity of azacrown ion exchanger was 0.89 meq/g dry resin. The heavier isotopes of magnesium were enriched in the resin phase, while the lighter isotopes were enriched in the solution phase. The hydration effect is less than the complexation and isotope mass effects. The single stage separation factor was determined according to the method of Glueckauf from the elution curve and isotopic assays. The separation factors of 24Mg(2+)-25Mg(2+), 24Mg(2+)-26Mg(2+), and 25Mg(2+)-26Mg(2+) were 1.012, 1.023, and 1.011, respectively.

Sensory and associated reactions to mineral dusts: sodium borate, calcium oxide, and calcium sulfate.

Occupational exposure limits (OELs) for irritant dusts have had no quantifiable bases. This study (1) charted chemosensory feel, denoted chemesthesis here, to dusts of calcium oxide (1 to 5 mg/m(3)), sodium tetraborate pentahydrate [sodium borate] (5 to 40 mg/m(3)), and calcium sulfate (10 to 40 mg/m(3)); (2) examined correlates of the chemesthetic sensations; and (3) sought to illuminate the basis for potency. Twelve screened men exercised against a light load while they breathed air in a dome fed with controlled levels of dust for 20 min. Measured parameters included nasal resistance, nasal secretion, minute ventilation, heart rate, blood oxygenation, mucociliary transport time, and chemesthetic magnitude, calibrated to pungency of carbon dioxide. Subjects registered time-dependent feel from exposures principally in the nose, secondarily in the throat, and hardly in the eyes. Calcium oxide had the greatest potency, followed by sodium borate, with calcium sulfate a distant third. Of the physiological parameters, amount of secretion showed the best association with chemesthetic potency. That measure, as well as mucociliary transport time and minute ventilation, went into calculation of mass of dust dissolved into mucus. The calculations indicated that the two alkaline dusts increased in equal molar amounts with time. At equal molar concentrations, they had, to a first approximation, equal chemesthetic magnitude. On the basis of mass concentration in air or dissolved into mucus, calcium oxide and sodium borate differed in potency by a factor just above five, equal to the difference in their molecular weights. This relationship could inform the setting of OELs for a critical effect of irritation.