One-Pot Synthesis of Diarylamines from Two Aromatic Amines via Oxidative Dearomatization–Imino Exchange–Reductive Aromatization

A one-pot synthetic strategy for diarylamines using only aromatic amines as starting materials has been developed. This method involved a PhI(OAc)₂-induced oxidative dearomatization of <i>N</i>-sulfonyl protected para-substituted anilines, a Bi(OTf)₃-catalyzed imino exchange reaction between <i>N</i>-sulfonyl cyclohexadienimines and aromatic amines, and a CF₃COOH/Zn mediated reductive aromatization of the resulting <i>N</i>-aryl cyclohexadienimines

Tel1 and Rif2 Regulate MRX Functions in End-Tethering and Repair of DNA Double-Strand Breaks

<div><p>The cellular response to DNA double-strand breaks (DSBs) is initiated by the MRX/MRN complex (Mre11-Rad50-Xrs2 in yeast; Mre11-Rad50-Nbs1 in mammals), which recruits the checkpoint kinase Tel1/ATM to DSBs. In <i>Saccharomyces cerevisiae</i>, the role of Tel1 at DSBs remains enigmatic, as <i>tel1</i>Δ cells do not show obvious hypersensitivity to DSB-inducing agents. By performing a synthetic phenotype screen, we isolated a <i>rad50-V1269M</i> allele that sensitizes <i>tel1</i>Δ cells to genotoxic agents. The MR^V1269MX complex associates poorly to DNA ends, and its retention at DSBs is further reduced by the lack of Tel1. As a consequence, <i>tel1</i>Δ <i>rad50-V1269M</i> cells are severely defective both in keeping the DSB ends tethered to each other and in repairing a DSB by either homologous recombination (HR) or nonhomologous end joining (NHEJ). These data indicate that Tel1 promotes MRX retention to DSBs and this function is important to allow proper MRX-DNA binding that is needed for end-tethering and DSB repair. The role of Tel1 in promoting MRX accumulation to DSBs is counteracted by Rif2, which is recruited to DSBs. We also found that Rif2 enhances ATP hydrolysis by MRX and attenuates MRX function in end-tethering, suggesting that Rif2 can regulate MRX activity at DSBs by modulating ATP-dependent conformational changes of Rad50.</p></div

Rif2 is recruited to DNA ends, and its lack enhances MRX and MR^V1269MX association to the DSB.

<p>(A, B) ChIP analysis. Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero. Relative fold enrichment of Mre11-Myc fusion protein at the indicated distances from the HO cleavage site was determined after ChIP with anti-Myc antibodies and subsequent qPCR analysis. Plotted values are the mean values with error bars denoting s.d. (<i>n</i> = 3). (C) Flag-tagged Tel1 (50 ng) was incubated with MR^V1269MX (100 ng) in the absence or presence of Rif2 (100 ng), and protein complexes were captured by anti-Flag resin and followed by immunoblotting analysis. (D) Rif2 (150, 300 and 600 nM) was incubated with ³²P-labeled 100-bp dsDNA (10 nM) in the presence of ATP. In lane 5, the reaction mixture was deproteinized with SDS and proteinase K (PK) prior to analysis. Plotted values are the mean value with error bars denoting s.d. (<i>n</i> = 3). (E) ChIP analysis. As in (A), but showing Rif2 recruitment at the HO-induced DSB.</p

The lack of Tel1 impairs MRX and MR^V1269MX association to a DSB.

<p>(A–D) ChIP analysis. Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero. Relative fold enrichment of the indicated fusion proteins at the indicated distances from the HO cleavage site was determined after ChIP with anti-HA or anti-Myc antibodies and subsequent qPCR analysis. Plotted values are the mean values with error bars denoting s.d. (<i>n</i> = 3). (E) Western blot with anti-HA or anti-Myc antibodies of extracts used for the ChIP analysis shown in (A–D). The same amount of protein extracts was separated on a SDS-PAGE and stained with Coomassie Blue (loading control).</p

<i>rad50-V1269M tel1</i>Δ mutant cells are defective in DSB repair by HR.

<p>(A) System to detect ectopic recombination. HO generates a DSB at a <i>MAT</i>a DNA sequence inserted on chromosome V, while the homologous <i>MAT</i>a-<i>inc</i> region on chromosome III cannot be cut by HO and is used as a donor for HR-mediated repair, which can generate both noncrossover (NCO) and crossover (CO) products. E, EcoRI. (B) Exponentially growing YEPR cell cultures were transferred to YEPRG at time zero. Southern blot analysis of EcoRI-digested genomic DNA with the <i>MAT</i>a probe depicted in A. (C) Densitometric analysis of CO versus NCO repair bands at the indicated times after HO induction (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002387#sec014" target="_blank">Materials and Methods</a>). (D) Mating type switching. Exponentially growing YEPR <i>MAT</i>a cell cultures (raf) were transferred to YEPRG to induce HO. After 30 min (gal), cells were transferred to YEPD to allow mating type switching. StyI-BamHI-digested genomic DNA prepared at the indicated times after glucose addition was subjected to Southern blot analysis with a <i>MAT</i>a probe. (E) Densitometric analysis of the <i>MAT</i>α product band signals (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002387#sec014" target="_blank">Materials and Methods</a>). Plotted values are the mean value with error bars denoting s.d. (<i>n</i> = 3). * indicates cross hybridization signals.</p

Rif2 enhances MRX ATPase activity.

<p>(A–C) The ATPase activity of wild-type MRX, MR^V1269MX and MR complexes was determined in the absence or presence of Rif2. Plotted values are the mean value with error bars denoting s.d. (<i>n</i> = 3). (D) Exponentially growing cultures were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin, or MMS. (E) Model for the roles of Tel1 and Rif2 in controlling MRX function at DSBs. In wild-type cells (left), Tel1 enhances MRX function by promoting MRX association to DSBs (black arrows). Proper MRX accumulation to DNA increases the number of productive intercomplex interactions between hook domains to maintain DNA strands in close proximity. Rif2 inhibits MRX accumulation at DSBs by counteracting MRX-Tel1 interaction (black bar-headed line) and enhances MRX ATPase activity (red arrows). In <i>tel1</i>Δ <i>rad50-V1269M</i> cells (middle), the reduced amount of MR^V1269MX bound at the DSB severely impairs end-tethering, HR and NHEJ. In <i>rif2</i>Δ <i>tel1</i>Δ <i>rad50-V1269M</i> cells (right), the lack of Rif2 restores end-tethering, HR and NHEJ by increasing the time spent by MRX in the ATP-bound state. The MR^V1269MX complex is indicated in grey.</p

Additional file 1 of CHRNB2 represses pancreatic cancer migration and invasion via inhibiting β-catenin pathway

Additional file 1: Figure S1. The expression conditions between tumor and paired normal pancreas among 13 genes in pancreatic cancer patients. A-M Compared to the paired normal pancreas, tumor tissue of most pancreatic cancer patients in GSE15471 has lower CHRNB2 expression. (**: P < 0.01; ***: P < 0.001)

Additional file 2 of CHRNB2 represses pancreatic cancer migration and invasion via inhibiting β-catenin pathway

Additional file 2: Figure S2. Flow cytometry showed that CHRNB2 arrested pancreatic cancer cells in the G1 phase. A Cell cycle flow chart of T3M4 with CHRNB2 overexpression. B Cell cycle histogram including G1, S, and G2 stages of T3M4 with CHRNB2 overexpression. (**: P < 0.01; ***: P < 0.001)

The <i>rad50-V1269M</i> mutation sensitizes <i>tel1</i>Δ cells to DNA damaging agents.

<p>(A) Multiple sequence alignment across seven organisms of Rad50 D-loop (yellow), H-loop (blue) and adjacent sequences. The valine residue substituted by methionine in Rad50-V1269M mutant is indicated by an asterisk. Abbreviations: A, Walker A; Q, Q-loop; S, Signature; B, Walker B; D, D-loop; H, H-loop; <i>Sc</i>, <i>Saccharomyces cerevisiae</i>; <i>Hs</i>, <i>Homo sapiens</i>; <i>Mm</i>, <i>Mus musculus</i>; <i>At</i>, <i>Arabidopsis thaliana</i>; <i>Dm</i>, <i>Drosophila melanogaster</i>; <i>Sp</i>, <i>Schizosaccharomyces pombe</i>; <i>Pf</i>, <i>Pyrococcus furiosus</i>. (B, C) Exponentially growing cultures were serially diluted (1:10) and each dilution was spotted out onto yeast extract peptone dextrose (YEPD) plates with or without CPT, phleomycin or methyl methanesulfonate (MMS) at the indicated concentrations.</p

The <i>rad50-V1269M</i> mutation impairs Rad50 ATPase and binding to DNA in vitro.

<p>(A) Purified Rad50, Rad50-V1269M, MRX, and MR^V1269MX, 1 μg each, were analyzed by SDS-PAGE and stained with Coomassie Blue. (B) The ATPase activity of wild-type MRX and mutant MR^V1269MX complexes was determined as described in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002387#pbio.1002387.ref027" target="_blank">27</a>]. Plotted values are the mean value with error bars denoting standard deviation (s.d.) (<i>n</i> = 3). (C) Rad50 and Rad50-V1269M proteins (50, 100, and 200 nM) were incubated with ³²P-labeled DNA (10 nM) in the presence of ATP. In lane 5 and 9, the reaction mixture was deproteinized with SDS and proteinase K (PK) prior to analysis. Plotted values are the mean value with error bars denoting s.d. (<i>n</i> = 3). (D) MRX and MR^V1269MX complexes (10, 20, and 40 nM) were treated as in (C).</p