Global fitness profiling of fission yeast deletion strains by barcode sequencing

Han, Tian Xu; Xu, Xingya; Zhang, Meijun; Peng, Xiaojue; Du, Li-Lin

doi:10.1186/gb-2010-11-6-r60

Cited by 89 publications

(104 citation statements)

References 70 publications

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“…However, how HU kills wild-type cells with a functional DRC is less understood. Although the DNA damage generated at HU-treated forks is believed to play an important role in the cell-killing process, recent studies suggest that HU may generate oxidative stress in Escherichia coli (Davies et al 2009;Foti et al 2012) and in S. cerevisiae (Chang et al 2002;Han et al 2010;Rowe et al 2012). However, it remains unclear whether this cell-killing mechanism is conserved in all eukaryotic organisms and how this mechanism is related to the cytokinesis arrest discovered in this study.…”

Section: Discussionmentioning

confidence: 54%

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

Singh

Alter

2016

Genetics

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Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood. While screening for new fission yeast mutants that are sensitive to replication stress, we identified a novel mutation in the erg11 gene encoding the enzyme sterol-14a-demethylase in the ergosterol biosynthesis pathway that dramatically sensitizes the cells to chronic HU treatment. Surprisingly, HU mainly arrests the erg11 mutant cells in cytokinesis, not in S phase. Unlike the reversible S phase arrest in wild-type cells, the cytokinesis arrest induced by HU is relatively stable and occurs at low doses of the drug, which likely explains the remarkable sensitivity of the mutant to HU. We also show that the mutation causes sterol deficiency, which may predispose the cells to the cytokinesis arrest and lead to cell death. We hypothesize that in addition to the RNR, HU may have a secondary unknown target(s) inside cells. Identification of such a target(s) may greatly improve the chemotherapies that employ HU or help to expand the clinical usage of this drug for additional pathological conditions.

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Section: Discussionmentioning

confidence: 54%

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

Singh

Alter

2016

Genetics

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“…Specifically, Dbl2 is needed for cellular resistance to the topoisomerase I poison camptothecin, forms DNA damageinduced foci, and is needed for the optimal recruitment of Fml1 to DNA damage (Han et al 2010;Yu et al 2013). Also, the meiotic chromosome segregation phenotype of the dbl2Δ mutant suggests that the Dbl2 protein plays a role in meiotic HR.…”

Section: Discussionmentioning

confidence: 99%

Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1

et al. 2016

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We identified Mte1 (Mph1-associated telomere maintenance protein 1) as a multifunctional regulator of Saccharomyces cerevisiae Mph1, a member of the FANCM family of DNA motor proteins important for DNA replication fork repair and crossover suppression during homologous recombination. We show that Mte1 interacts with Mph1 and DNA species that resemble a DNA replication fork and the D loop formed during recombination. Biochemically, Mte1 stimulates Mph1-mediated DNA replication fork regression and branch migration in a model substrate. Consistent with this activity, genetic analysis reveals that Mte1 functions with Mph1 and the associated MHF complex in replication fork repair. Surprisingly, Mte1 antagonizes the D-loop-dissociative activity of Mph1-MHF and exerts a procrossover role in mitotic recombination. We further show that the influence of Mte1 on Mph1 activities requires its binding to Mph1 and DNA. Thus, Mte1 differentially regulates Mph1 activities to achieve distinct outcomes in recombination and replication fork repair.

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“…Previous studies have sequence barcoded up to 6,200 yeast (39,40) or viral (39,40) strains for competition experiments and analyzed the barcodes by deep sequencing (Bar-seq) or microarrays. As we were competing a relatively small number of viral strains (nine), we identified the barcodes using an approach based on the principles of serial analysis of gene expression (SAGE) (41).…”

Section: Discussionmentioning

confidence: 99%

Evasion of Superinfection Exclusion and Elimination of Primary Viral RNA by an Adapted Strain of Hepatitis C Virus

Webster

Ott

Greene

2013

J Virol

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Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequencebarcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/ UC) tract of the 3= untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo. In superinfection exclusion, a cell productively infected with a specific virus becomes resistant to infection with a homologous virus. This process has been described for several viruses, including human immunodeficiency virus (HIV) (1), Sindbis virus (2), duck hepatitis B virus (3), and citrus tristeza virus (4), in a broad range of hosts. Cells infected with or actively replicating hepatitis C virus (HCV) also become refractory to further HCV infection (5, 6). The superinfection block occurs at the level of translation or replication of the incoming secondary viral RNA (5-7), which is similar to the case of the related pestivirus bovine diarrhea virus (8). The exact mechanism is unclear. Since HCV RNA levels quickly plateau in infected cells in vitro, a superinfection block at the level of RNA translation or replication suggests that access to the necessary host factor(s) is rate limiting. This has clear implications for HCV treatment. By definition, a rate-limiting host factor is critical for the replication of the virus and may be a unique target in future therapies.Superinfection exclusion itself also has clear implications for treating HCV infection. Since viral recombination requires a host cell to be productively replicating two genomes, superinfection exclusion would be expected to effectively prevent viral recombination. If, howeve...

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Global fitness profiling of fission yeast deletion strains by barcode sequencing

Cited by 89 publications

References 70 publications

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

Differential regulation of the anti-crossover and replication fork regression activities of Mph1 by Mte1

Evasion of Superinfection Exclusion and Elimination of Primary Viral RNA by an Adapted Strain of Hepatitis C Virus

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