THE WEIMAR REPUBLIC: BEYOND CONVENTIONAL VISION. Historiographical debate essays.

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Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs’ structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group’s contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb’s characterization workflows

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. We show that Tel1, the orthologue of human ATM kinase, stabilizes CPT-induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1-lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated Δ cells without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, Δ and nuclease-deficient cells and relieves the hypersensitivity of Δ cells to CPT Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1-mediated reversal in the presence of CPT

Sae2 Function at DNA Double-Strand Breaks Is Bypassed by Dampening Tel1 or Rad53 Activity

<div><p>The MRX complex together with Sae2 initiates resection of DNA double-strand breaks (DSBs) to generate single-stranded DNA (ssDNA) that triggers homologous recombination. The absence of Sae2 not only impairs DSB resection, but also causes prolonged MRX binding at the DSBs that leads to persistent Tel1- and Rad53-dependent DNA damage checkpoint activation and cell cycle arrest. Whether this enhanced checkpoint signaling contributes to the DNA damage sensitivity and/or the resection defect of <i>sae2</i>Δ cells is not known. By performing a genetic screen, we identify <i>rad53</i> and <i>tel1</i> mutant alleles that suppress both the DNA damage hypersensitivity and the resection defect of <i>sae2</i>Δ cells through an Sgs1-Dna2-dependent mechanism. These suppression events do not involve escaping the checkpoint-mediated cell cycle arrest. Rather, defective Rad53 or Tel1 signaling bypasses Sae2 function at DSBs by decreasing the amount of Rad9 bound at DSBs. As a consequence, reduced Rad9 association to DNA ends relieves inhibition of Sgs1-Dna2 activity, which can then compensate for the lack of Sae2 in DSB resection and DNA damage resistance. We propose that persistent Tel1 and Rad53 checkpoint signaling in cells lacking Sae2 increases the association of Rad9 at DSBs, which in turn inhibits DSB resection by limiting the activity of the Sgs1-Dna2 resection machinery.</p></div

Rad53-kd and Tel1-kd prevent Rad9 association at DSBs.

<p>(A) Exponentially growing YEPR cell cultures of JKM139 derivative strains were arrested in G2 with nocodazole and transferred to YEPRG in the presence of nocodazole. Recruitment of Rad9-HA at the indicated distance from the HO-cut was determined by ChIP and qPCR. In all diagrams, the ChIP signals were normalized for each time point to the amount of the corresponding input signal. The mean values are represented with error bars denoting s.d. (n = 3). (B) As in (A), but showing Sgs1-HA binding. (C) As in (A). All strains carried also the deletion of <i>HTA2</i> gene. (D) As in (A), but showing γH2A binding. (E) Model for the role of Sae2 at DSBs. (Left) Sae2 activates the Mre11 endonuclease activity to incise the 5’ strand. Generation of the nick allows bidirectional processing by Exo1/Sgs1-Dna2 in the 5’-3’ direction from the nick and MRX in the 3’ to 5’ direction toward the DSB ends. Ku and Rad9 inhibit DSB resection by limiting Exo1 and Sgs1-Dna2, respectively. (Middle) The absence of Sae2 impairs the MRX nuclease activity (non functional MRX nuclease is in grey). As a consequence, the endonucleolytic cleavage of the 5’ strand does not occur and resection is carried out by Exo1 and Dna2-Sgs1 that degrade the 5’ strands from the DSB ends. Impairment of Mre11 nuclease activity also causes increased MRX association at the DSB, which leads to enhanced Tel1-dependent Rad53 activation. Tel1 and Rad53 activities limit DSB resection from the DSB end (dashed arrow) by increasing the amount of DSB-bound Rad9, which inhibits Sgs1-Dna2 recruitment at DSBs. (Right) Impairments of Tel1 or Rad53 activity (non functional Tel1 and Rad53 are in grey) restore efficient resection in <i>sae2</i>Δ cells by relieving Rad9-mediated inhibition of Sgs1-Dna2. Restored DSB resection by Sgs1-Dna2 also reduces MRX persistence at the DSB.</p

The Rad53-H88Y and Tel1-N2021D bypass of Sae2 function is Sgs1-Dna2-dependent.

<p>(A, B) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS. (C-F) Meiotic tetrads were dissected on YEPD plates that were incubated at 25°C, followed by spore genotyping. (G) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates.</p

Rad53-H88Y and Tel1-N2021D suppress the hypersensitivity to genotoxic agents of <i>sae2</i>Δ cells.

<p>(A-D) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT, phleomycin or MMS.</p

The lack of Chk1 does not suppress the hypersensitivity to DNA damaging agents of <i>sae2</i>Δ cells.

<p>(A) Exponentially growing YEPR cultures of JKM139 derivative strains were transferred to YEPRG (time zero), followed by western blot analysis with anti-Rad53 antibodies. (B) YEPR G1-arrested cell cultures of JKM139 derivative strains were plated on galactose-containing plates (time zero). At the indicated time points, 200 cells for each strain were analyzed to determine the frequency of large budded cells (2 cells) and of cells forming microcolonies of 4 or more than 4 cells. (C) Exponentially growing cells were serially diluted (1:10) and each dilution was spotted out onto YEPD plates with or without CPT and phleomycin. (D, E) DSB resection. YEPR exponentially growing cultures of JKM139 derivative cells were arrested in G2 with nocodazole and transferred to YEPRG in the presence of nocodazole at time zero. Gel blots of SspI-digested genomic DNA separated on alkaline agarose gel were hybridized with a single-stranded RNA probe that anneals to the unresected strand on one side of the break. 5’-3’ resection progressively eliminates SspI sites, producing larger SspI fragments (r1 through r6) detected by the probe.</p

Rad53-H88Y and Tel1-N2021D suppress the checkpoint shut off defect of <i>sae2</i>Δ cells.

<p>(A) G1-arrested cell cultures of JKM139 derivative strains were plated on galactose-containing plates (time zero). At the indicated time points, 200 cells for each strain were analyzed to determine the frequency of large budded cells (2 cells) and of cells forming microcolonies of 4 or more than 4 cells. (B) Exponentially growing YEPR cultures of the strains in (A) were transferred to YEPRG (time zero), followed by western blot analysis with anti-Rad53 antibodies. (C) Protein extracts were analyzed by western blot with anti-HA or anti-Rad53 antibodies either directly (Total) or after Rad9-HA immunoprecipitation (IPs) with anti-HA antibodies. (D) Protein extracts from exponentially growing cells were analyzed by western blotting with anti-HA antibodies. The same amounts of protein extracts were separated by SDS-PAGE and stained with Coomassie as loading control. (E) Kinase assay was performed on equal amounts of anti-HA immunoprecipitates of protein extracts from cells either exponentially growing in YEPD or after treatment with 50μM CPT for 1 hour. All the immunoprecipitates were also subjected to western blot analysis using anti-HA antibodies. (F) Relative fold enrichment of Tel1-HA and Tel1-N2021D-HA compared to untagged Tel1 (no tag) at the indicated distance from the HO cleavage site was evaluated after ChIP with anti-HA antibodies and qPCR analysis. In all diagrams, the ChIP signals were normalized for each time point to the amount of the corresponding immunoprecipitated protein and input signal. The mean values are represented with error bars denoting s.d. (n = 3).</p