On the viability of Escherichia coli cells lacking DNA topoisomerase I

Copyright @ 2012 Stockum et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article is made available through the Brunel Open Access Publishing Fund.Background: Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I. Results: In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG. Conclusions: Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.The Medical Research Council (grant G0800970) and The Leverhulme Trust

Identification and characterisation of Ubiquitin Specific Protease 11 binding partners

Throughout the last decades, post-translational modification of proteins by ubiquitination has proved to be important for most cellular processes. The regulation depends on ubiquitin addition, which is orchestrated by a cascade of three enzymes, as well as ubiquitin removal, catalysed by deubiquitinating enzymes (DUBs). The human genome encodes nearly 100 DUBs of which the Ubiquitin Specific Proteases (USPs) are the largest of the five DUB families. This project focuses on USP11, which was previously thought to play a role in DNA damage response (DDR). In order to further understand the possible role for USP11 in DDR, we have identified several USP11 interacting proteins, namely PAM, SPRYD3 and RAE1. We show that SPRYD3 and RAE1 are ubiquitinated and also identify these proteins as USP11 substrates. The E3 ligase, PAM functions as a SCF-like complex with the F-box protein FBXO45. Our data shows strong similarities between this interaction and the interaction of PAM with SPRYD3, suggesting that the two proteins could form an alternative E3 ligase complex. Another USP11 interacting protein, RAE1, also interacts with PAM, which was verified in this study. RAE1 was identified as an RNA export factor, but was recently shown to be involved in regulation of the mitotic checkpoint in a complex with NUP98. Indeed, growth curves of RAE1 depleted U2OS cells show a significant lack in cell proliferation compared to control shRNA expressing cells. Surprisingly, a similar growth defect was observed in USP11 depleted cells. Ablation of RAE1 or USP11 allowed a small fraction of nocodazole arrested cells to proceed in the cell cycle, suggesting that the mitotic checkpoint is affected by the lack of either of these proteins. While further investigations are required to prove our hypotheses, the presented work identifies a variety of new substrates for USP11 and a new pathway it appears to regulate.Open Acces

RAE1 and USP11 are associated with the mitotic spindle.

<p>(<b>A</b>) Schematic representation of experimental workflow of mitotic spindle isolation. U2OS cells expressing indicated shRNAs were treated with nocodazole for 24h. Mitotic cells were washed off, released and treated briefly with taxol before harvesting. Spindle fractionation was done as described previously ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190513#pone.0190513.ref035" target="_blank">35</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190513#pone.0190513.ref036" target="_blank">36</a>], see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190513#sec009" target="_blank">Material and Methods</a>). The western blots shown are representative of the results obtained for three independent biological replicates. (<b>B</b>) Purified mitotic spindles were separated by SDS-PAGE (11%) and the indicated proteins were detected by western blot. Knock-down of USP11 does not affect mitotic spindle localization of RAE1. The numbers underneath the USP11 western blot indicate the normalized USP11 protein levels in each sample. (<b>C</b>) Purified mitotic spindles from U2OS cells transduced with the indicated shRNAs, were separated on a 7.5% SDS-PAGE gel and transferred onto nitrocellulose for Western blot detection. Representative western blots of protein knock-downs of the transductions used to quantify bi/multi-polar spindles in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190513#pone.0190513.g006" target="_blank">Fig 6</a>. Antibodies used are indicated to the right of the western blots. Positive controls for mitotic spindle isolation were included (ß-tubulin, DYNEIN, CENP-E, PLK1 and PP2A-Calpha) as well as the chromatin modifier, HDAC6, which was previously shown not to associate with mitotic spindles [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190513#pone.0190513.ref035" target="_blank">35</a>]. Mw markers are indicated to the left of the western blots. The numbers underneath the USP11 and NuMA western blots illustrate the normalized protein levels of USP11 and, respectively, NuMA. (<b>D</b>) USP11 does not influence RAE1 association with the mitotic spindle. The amount of RAE1 associated with mitotic spindles upon USP11 or RAE1 knock-down was quantified, normalized to the loading control and compared to cells treated with control shRNA (n = 4). Averages and SEM of four independent knock-down and fractionations are shown. P-values were calculated using the two-tailed paired t-test; compared to the control shRNA transduced cells, and are indicated as follows: ns: p > 0.05; **: 0.0001 < p < 0.001. (<b>E</b>) U2OS cells were treated with nocodazole for 24 h, the cells were carefully washed with pre-warmed PBS and treated with 5 μM MG132 for 90 min before fixation. Immunostaining of endogenous USP11, ß-tubulin and DNA was done as described in Materials and Methods.</p

USP11 deubiquitinates RAE1 and plays a key role in bipolar spindle formation

<div><p>Correct segregation of the mitotic chromosomes into daughter cells is a highly regulated process critical to safeguard genome stability. During M phase the spindle assembly checkpoint (SAC) ensures that all kinetochores are correctly attached before its inactivation allows progression into anaphase. Upon SAC inactivation, the anaphase promoting complex/cyclosome (APC/C) E3 ligase ubiquitinates and targets cyclin B and securin for proteasomal degradation. Here, we describe the identification of Ribonucleic Acid Export protein 1 (RAE1), a protein previously shown to be involved in SAC regulation and bipolar spindle formation, as a novel substrate of the deubiquitinating enzyme (DUB) Ubiquitin Specific Protease 11 (USP11). Lentiviral knock-down of USP11 or RAE1 in U2OS cells drastically reduces cell proliferation and increases multipolar spindle formation. We show that USP11 is associated with the mitotic spindle, does not regulate SAC inactivation, but controls ubiquitination of RAE1 at the mitotic spindle, hereby functionally modulating its interaction with Nuclear Mitotic Apparatus protein (NuMA).</p></div

Ablation of USP11 increases ubiquitination of RAE1 on the mitotic spindle.

<p>(<b>A</b>) Schematic representation of the mitotic spindle fractionation followed by NiNTA pull-down of His₆-ubiquitinated proteins. (<b>B</b>) U2OS cells stably expressing His₆-ubiquitin were transduced with a control shRNAs or shRNAs targeting USP7 or USP11 transcripts. A U2OS cell line that lacks ectopic expression of His₆-ubiquitin was used as negative control. After 24h treatment with nocodazole mitotic cells were harvested by shake off, released for 90 min in complete medium supplemented with 5 μM MG132, and treated briefly with taxol (3 min) before harvesting, fractionation was done as shown in (<b>A</b>). Samples (input and NiNTA pull-down) were separated on a 7.5% SDS-PAGE gel before transfer onto nitrocellulose membrane. Antibodies used are indicated to the right of each blot; *, indicate non-specific bands.</p

Knock-down of USP11 restores bipolar spindle formation in NuMA shRNA transduced cells.

<p>(<b>A-H</b>) Representative images of U2OS cells expressing the indicated shRNAs released from nocodazole arrest for 90 min, immunostained for ß-tubulin. Z-stacks were projected into 1 image. The striped line indicates separate images. (<b>I</b>) Percentage of multipolar spindles observed in cells expressing the indicated shRNA. The total number of counted cells is indicated in the bars for each shRNA. (<b>J</b>) Percentage of multipolar spindles expressing the indicated shRNA in combination with knock-down of NuMA. The total number of counted cells is indicated in the bars for each shRNA. Averages and SEMs are shown; ns: not significant, p > 0.05; * 0.01< p < 0.05; ** 0.001 < p < 0.01. The actual p-values for n biological replicates are, compared to control shRNA treated cells (n = 4): USP11sh1 (n = 4, p-value = 0.0036); USP7sh1 (n = 3, p-value = 0.0070); RAE1sh3 (n = 3, p-value = 0.0155); NuMAsh2 + control shRNA (n = 3, p-value = 0.0014); and compared to NuMAsh2 + ctrl shRNA treated cells: USP7sh1 + NuMAsh2 (n = 2, p-value = 0.457); NuMAsh2 + RAE1sh3 (n = 3, p-value = 0.0043), and NuMAsh2 + USP11sh1 (n = 3, p-value = 0.0317).</p

U2OS cell proliferation upon USP11 or RAE1 knock-down.

<p>U2OS cell proliferation measured by MTT. (<b>A</b>) Knock-down of RAE1 or USP11 significantly reduces cell proliferation compared to control shRNA transduced cells (p = 0.0004; p = 0.0004, respectively). (<b>C</b>) MTT cell viability assay measuring cell growth of U2OS cells transduced with control, USP11 sh1 (p = 0.003) or USP11 sh5 (p = 0.0088) shRNA. (<b>E</b>) Back-complementation with RAE1 rescues RAE1 shRNA reduced cell proliferation. RAE1shRNA transduced U2OS cells, back-complemented with shRNA resistant RAE1 (RAE1 shR) partially restores growth rates to the control cells (p = 0.0021 for RAE1sh3 + Flag control compared to RAE1sh3 back-complemented with RAE1). (<b>B</b>, <b>D and F</b>) Western blots illustrating respective protein knock-downs, these are representative for 3 biological replicates. Antibodies used are indicated to the right of each panel. Where blots have been separated by a white line, this indicates that lanes from the western blot, irrelevant to the experiment shown were removed. In panels (<b>B</b> and <b>D</b>) the numbers shown underneath the USP11 and RAE1 western blots indicate the respective normalized protein levels upon knock-down with the different shRNAs compared to control shRNA treated cells. Molecular weight markers are indicated to the left of each western blot. Averages and SEM of three independent transductions and growth curves are shown. P-values were calculated using the two-tailed paired t-test; compared to the control shRNA transduced cells, and are indicated as follows: **: 0.001 < p < 0.01; ***: p < 0.001.</p

Knock-down of USP11 or RAE1 does not induce premature SAC inactivation.

<p>(<b>A</b>) Schematic representation of experimental workflow. U2OS cells expressing shRNA targeting USP11 or RAE1 transcripts were incubated for 24h with nocodazole. Mitotic cells were shaken off, washed and released from nocodazole arrest. Samples were taken at indicated time points post-release. (<b>B</b>) Western blot verification of USP11 and RAE1 knock-down. (<b>C</b>) Western blots illustrating the changes in cyclin B1 and securin protein levels upon knock-down of USP11 or RAE1 compared to control shRNA transduced cells. APC3 dephosphorylation was used as a marker for metaphase to anaphase progression. Antibodies used are indicated to the right of the western blots, molecular weight markers are indicated to the left of the western blot. shRNAs, and time upon release from nocodazole (min) is shown above the western blots. The data shown is representative of 3 biological replicates.</p

USP11 does not regulate RAE1 protein levels in cells.

<p>RAE1 protein levels in the total cell lysate were quantified upon USP11 (n = 5) or RAE1 knock-down (n = 6), normalized to the loading control and compared to the RAE1 protein levels in control shRNA treated cells. Averages and SEM of 5–6 independent transductions are shown. P-values were calculated using the two-tailed paired t-test; compared to the control shRNA transduced cells, and are indicated as follows: ns: not significant, ****: p<0.0001.</p