Rice (Oryza sativa) TIR1 and 5′adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

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Replication Factor C3 of Schizosaccharomyces pombe, a Small Subunit of Replication Factor C Complex, Plays a Role in Both Replication and Damage Checkpoints

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.3991 ◽

1999 ◽

Vol 10 (12) ◽

pp. 3991-4003 ◽

Cited By ~ 44

Author(s):

Midori Shimada ◽

Daisuke Okuzaki ◽

Seiji Tanaka ◽

Takahiro Tougan ◽

Katsuyuki K. Tamai ◽

...

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Uv Irradiation ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Replication Factor C ◽

Replication Factor ◽

Factor C

We report here the isolation and functional analysis of therfc3 + gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3 + gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired inrfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 inrfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3 + is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor.

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The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.1.65 ◽

1996 ◽

Vol 142 (1) ◽

pp. 65-78 ◽

Cited By ~ 7

Author(s):

Michael A McAlear ◽

K Michelle Tuffo ◽

Connie Holm

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Dna Replication ◽

Uv Radiation ◽

Large Subunit ◽

Restrictive Temperature ◽

Replication Factor C ◽

Replication Factor ◽

Factor C

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

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Auxinic Herbicide Resistance May Be Modulated at the Auxin-Binding Site in Wild Mustard (Sinapis arvensis L.): A Light Scattering Study

Pesticide Biochemistry and Physiology ◽

10.1006/pest.1999.2450 ◽

2000 ◽

Vol 66 (1) ◽

pp. 41-48 ◽

Cited By ~ 19

Author(s):

Satish Deshpande ◽

J.Christopher Hall

Keyword(s):

Light Scattering ◽

Binding Site ◽

Herbicide Resistance ◽

Sinapis Arvensis ◽

Wild Mustard ◽

Auxin Binding ◽

Scattering Study

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Genome-wide distribution of DNA replication origins at A+T-rich islands in Schizosaccharomyces pombe

EMBO Reports ◽

10.1038/sj.embor.embor7400008 ◽

2003 ◽

Vol 4 (11) ◽

pp. 1048-1053 ◽

Cited By ~ 3

Author(s):

Mónica Segurado ◽

Alberto de Luis ◽

Francisco Antequera

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Wide Distribution ◽

Replication Origins ◽

Genome Wide ◽

Dna Replication Origins

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Upstream – News in Genomics

Comparative and Functional Genomics ◽

10.1002/cfg.172 ◽

2002 ◽

Vol 3 (3) ◽

pp. 221-225

Keyword(s):

Saccharomyces Cerevisiae ◽

Ovarian Cancer ◽

Oryza Sativa ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Proteomic Analysis ◽

Large Scale ◽

Protein Complexes ◽

The Other ◽

Blood Samples

In recent months a bumper crop of genomes has been completed, including the fission yeast (Schizosaccharomyces pombe) and rice (Oryza sativa). Two large-scale studies ofSaccharomyces cerevisiaeprotein complexes provided a picture of the eukaryotic proteome as a network of complexes. Amongst the other stories of interest was a demonstration that proteomic analysis of blood samples can be used to detect ovarian cancer, perhaps even as early as stage I.

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Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2790-2801.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2790-2801 ◽

Cited By ~ 31

Author(s):

James F. Theis ◽

Carol S. Newlon

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Binding Site ◽

Binding Sites ◽

Origin Recognition Complex ◽

Replication Origins ◽

Discrete Site ◽

Origin Function ◽

Single Binding Site ◽

Origin Recognition

ABSTRACT While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins inSaccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

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Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.86 ◽

1996 ◽

Vol 16 (1) ◽

pp. 86-93 ◽

Cited By ~ 45

Author(s):

R Kovelman ◽

P Russell

Keyword(s):

Dna Replication ◽

Schizosaccharomyces Pombe ◽

Replication Checkpoint ◽

Activation Assay ◽

M Phase ◽

Dna Replication Checkpoint ◽

High Level ◽

Tyrosyl Phosphorylation ◽

In Cells

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

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