Replication Checkpoint Enforced by Kinases Cds1 and Chk1
10.1126/science.280.5365.909
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Cdc2, the kinase that induces mitosis, is regulated by checkpoints that couple mitosis to the completion of DNA replication and repair. The repair checkpoint kinase Chk1 regulates Cdc25, a phosphatase that activates Cdc2. Effectors of the replication checkpoint evoked by hydroxyurea (HU) are unknown. Treatment of fission yeast with HU stimulated the kinase Cds1, which appears to phosphorylate the kinase Wee1, an inhibitor of Cdc2. The protein kinase Cds1 was also required for a large HU-induced increase in the amount of Mik1, a second inhibitor of Cdc2. HU-induced arrest of cell division was abolished in cds1 chk1 cells. Thus, Cds1 and Chk1 appear to jointly enforce the replication checkpoint.

Bibliography

Boddy, M. N., Furnari, B., Mondesert, O., & Russell, P. (1998). Replication Checkpoint Enforced by Kinases Cds1 and Chk1. Science, 280(5365), 909–912.

Authors 4
  1. Michael N. Boddy (first)
  2. Beth Furnari (additional)
  3. Odile Mondesert (additional)
  4. Paul Russell (additional)
References 23 Referenced 258
  1. 10.1016/0092-8674(92)90586-2
  2. 10.1126/science.274.5293.1664
  3. Enoch T., Nurse P., Cell60, 665 (1990). (10.1016/0092-8674(90)90669-6) / Cell by Enoch T. (1990)
  4. Lundgren K., et al., ibid64, 1111 (1991). / ibid by Lundgren K. (1991)
  5. P. Jin, Y. Gu, D. O. Morgan, J. Cell Biol. 134, 963 (1996); Blasina A., Paegle S., McGowan C. H., Mol. Biol. Cell 8, 1013 (1997). (10.1091/mbc.8.6.1013) / Mol. Biol. Cell by Blasina A. (1997)
  6. Rhind N., Furnari B., Russell P., Genes Dev.11, 504 (1997). (10.1101/gad.11.4.504) / Genes Dev. by Rhind N. (1997)
  7. 10.1126/science.277.5331.1495
  8. Y. Sanchez et al. ibid. p. 1497; C.-Y. Peng et al. ibid. p. 1501.
  9. N. Walworth, S. Davey, D. Beach, Nature 363, 368 (1993); al-Khodairy F., et al., Mol. Biol. Cell 5, 147 (1994). (10.1091/mbc.5.2.147) / Mol. Biol. Cell by al-Khodairy F. (1994)
  10. N. Rhind and P. Russell unpublished data.
  11. M. N. Boddy and P. Russell unpublished data.
  12. Aligue R., Wu L., Russell P., J. Biol. Chem.272, 13320 (1997). (10.1074/jbc.272.20.13320) / J. Biol. Chem. by Aligue R. (1997)
  13. A polymerase chain reaction (PCR) fragment was made encoding amino acids 11 to 152 of Wee1 incorporating a 5′ Nde I site and 3′ Not I site–stop codon. This fragment was cloned into a modified pGEX vector and transformed into the Escherichia coli BL21 (DE3) expression host (22). GST:Wee1 152 protein expression was induced by the addition of isopropyl-β- d -thiogalactopyranoside (1 mM) to exponentially growing cells at an absorbance at 600 nm ( A 600 ) of 0.6 in LB media plus ampicillin (50 μg/ml) at 372°C. After 2 hours at 37°C cells were collected by centrifugation and stored at −70°C in portions (200-μl pellet volume). Cells were lysed in 600 μl of lysis buffer [50 mM tris (pH 8) 150 mM NaCl 5 mM EDTA 10% glycerol 0.1% NP-40 leupeptin-aprotinin-pepstatin (5 μg/ml) and 1 mM phenylmethylsulfonyl fluoride] by sonication and centrifuged at 13 000 g for 10 min at 4°C. GSH-Sepharose (50-μl bead volume) was added to the supernatant and incubated at 4°C for 30 min. The beads were then washed three times with 1 ml of lysis buffer. Key experiments involving the GST:Wee1 70 truncation product were replicated with a precise fusion of GST and amino acids 1 to 72 of Wee1.
  14. Strains of the following genotypes were used: PR109, wild type; NB2117, cds1::ura4 +; NB2118,cds1:2HA6His:ura4 +; NR1826,rad3::ura4 +; OM591,cdc22-M45; NR1593,rad1::ura4 +; NR1592,chk1::ura4 +; BF1758,nmt1:GST:chk1:leu1 +; BF1916,nmt1:GST:cds1:leu1 +; OM2173,mik1:2HA6His:ura4 +; OM2183,mik1:2HA6His:ura4 + cds1::ura4 +; PR712,mik1::ura4 +; and BF2115,cds1::ura4 + chk1::ura4 +. All strains wereleu1-32 ura4-D18. All nmt1 promoter constructs were integrated at the leu1-32 locus. Growth media and general methods for S. pombe have been described [S. Moreno, A. Klar, P. Nurse, Methods Enzymol. 194, 795 (1991)]. Unless otherwise indicated, yeast cultures were grown in YES medium at 30°C. YES consists of glucose, yeast extract, and amino acid supplements. HU was used at a concentration of 12 mM. Purification of GST fusion proteins and hexahis-tagged proteins expressed in S. pombe, immunoblotting, and kinase assays were performed as described [Shiozaki K., Russell P., Nature 378, 739 (1995)]. (10.1038/378739a0) / Nature by Shiozaki K. (1995)
  15. Fernandez-Sarabia M., McInerny C., Harris P., Gordon C., Fantes P., Mol. Gen. Genet.238, 241 (1993). / Mol. Gen. Genet. by Fernandez-Sarabia M. (1993)
  16. Murakami H., Okayama H., Nature374, 817 (1995). (10.1038/374817a0) / Nature by Murakami H. (1995)
  17. To tag genomic cds1 + with a sequence encoding two copies of the HA epitope and hexahistidine we made a Pst I–Not I fragment having nucleotides 205 to 1465 of the cds1 + open reading frame by PCR. This fragment was introduced into pRIP42-Spc1HA6H after digestion with Pst I and Not I enzymes (22). The construct was linearized with Nhe I and transformed into PR109. Integration and tagging were confirmed by Southern (DNA) hybridization and immunoblotting with function of Cds1 HAHIS confirmed by lack of HU sensitivity. The nmt1:GST:cds1 + construct was prepared as described for the nmt1:GST:chk1 + construct (7).
  18. The in vivo interaction between GST:Cds1 and Wee1 shown in Fig. 2 D did not require HU treatment presumably because GST:Cds1 was overexpressed.
  19. Cells were grown in YES medium to an A 600 of 1.0 and then treated with HU (12 mM) for 90 min at 30°C. Synchronous cells were then obtained by centrifugal elutriation with a Beckman JE-5.0 elutriation rotor. These cells were diluted to an A 600 of 0.3 in YES containing 12 mM HU and grown at 30°C for a further 4 hours. Cells were scored for progression through mitosis by microscopic observation. HU sensitivity studies were carried out with cells grown to an A 600 of 0.3 in YES medium and then diluted to 13 000 cells/ml in YES media containing HU (12 mM) followed by growth at 30°C for a further 8 hours. Samples (75 μl) were taken at regular intervals plated onto YES plates and grown for 4 days at 30°C to determine survival.
  20. 10.1101/gad.12.3.382
  21. Enoch T., Carr A. M., Nurse P., ibid6, 2035 (1993). / ibid by Enoch T. (1993)
  22. Shiozaki K., Russell P., Methods Enzymol.283, 506 (1997). (10.1016/S0076-6879(97)83040-6) / Methods Enzymol. by Shiozaki K. (1997)
  23. We thank N. Rhind J.-M. Brondello C. McGowan and K. Shiozaki for their advice and the Scripps Cell Cycle Groups for their support and encouragement. Funded by NIH.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:37 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:34 p.m.)
Indexed 9 months, 1 week ago (Nov. 19, 2024, 10:43 a.m.)
Issued 27 years, 3 months ago (May 8, 1998)
Published 27 years, 3 months ago (May 8, 1998)
Published Print 27 years, 3 months ago (May 8, 1998)
Funders 0

None

@article{Boddy_1998, title={Replication Checkpoint Enforced by Kinases Cds1 and Chk1}, volume={280}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.280.5365.909}, DOI={10.1126/science.280.5365.909}, number={5365}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Boddy, Michael N. and Furnari, Beth and Mondesert, Odile and Russell, Paul}, year={1998}, month=may, pages={909–912} }