Multiple kinases inhibit origin licensing and helicase activation to ensure reductive cell division during meiosis
- Massachusetts Institute of Technology, United States
- Howard Hughes Medical Institute, United States
- Columbia University Medical Center, United States
Figures
Figure 1 with 3 supplements
Mcm2–7 loading onto replication origins is inhibited during the MI–MII transition.
(A) The DNA replication program and chromosome segregation program are uncoupled during the MI–MII transition. Relative CDK activity at various stages of the meiotic cell cycle are shown (Carlile …
-
Figure 1—source data 1
Raw values used for the quantification of Figure 1B and C.
- https://doi.org/10.7554/eLife.33309.006
Figure 1—figure supplement 1
Cells from Figure 1 proceeded synchronously through meiosis.
(A) Cell–cycle stage quantification for Figure 1B. (B) Cell–cycle stage quantification for Figures 1C and 2A. (C) Representative micrographs from a separate experiment showing the peak time point of …
Figure 1—figure supplement 2
Both ORC and Mcm2-7 associate specifically with origins of replication compared to non-origin DNA.
(A) Enrichment of DNA immunoprecipitated (as a percent of input) was compared at different origins of replication relative to non-origin control DNA (URA3 locus). For ORC, the G2 ChIP was compared …
Figure 1—figure supplement 3
Mcm2–7, Cdt1, and Orc1 proteins are present throughout meiosis.
(A) Top: Immunoblots showing that Mcm2, Mcm3, Mcm6, Mcm7, Cdt1, and Orc1 are present at constant levels throughout meiosis (strain yDP71). Pgk1 and Ponceau are shown as loading controls. Bottom: …
Figure 2 with 1 supplement
CDK-dependent inhibitory mechanisms are weakened during the MI–MII transition.
(A) Orc2 (strain yDP71) and (B) Orc6 (strain yDP120) are both transiently dephosphorylated during the MI–MII transition. ORC was detected by immunoblot during meiosis. A phosphorylation–dependent …
Figure 2—figure supplement 1
Cells from Figure 2 proceeded synchronously through meiosis.
(A) Cell–cycle stage quantification for Figure 2B. (B) Cell–cycle stage quantification for Figure 2C.
Figure 3 with 2 supplements
Ime2 is sufficient to inhibit helicase loading in vitro.
(A) Diagram of helicase–loading and OCCM–complex–formation assays. Origin–containing DNA (red) is bound to a magnetic bead. Origin bound ORC–Cdc6 complexes recruit Cdt1–Mcm2–7 heptamers to form the …
Figure 3—figure supplement 1
Ime2-AS inhibits helicase loading.
(A) Ime2–AS can inhibit Mcm2–7 loading to a similar extent as wild–type Ime2 (compare to Figure 3C). Top: Flowchart of experiment. Bottom: Helicase–loading assay at the indicated Ime2–AS …
Figure 3—figure supplement 2
Ime2 kinase activity correlates with inhibition of helicase loading.
(A) Increased Ime2 concentration causes increased phosphorylation of the helicase–loading proteins. Top: total protein (Krypton stain). Bottom: phosphorylated protein (modified with [γ-32P] ATP). (B)…
Figure 4 with 1 supplement
Ime2–phosphorylation of the Mcm2–7 complex intrinsically inhibits its loading onto replication origins.
(A) Ime2 can phosphorylate Cdc6, ORC, Cdt1 and Mcm2–7 in vitro. Buffer control (lanes 1, 3, and 5) or 50 nM Ime2 (lanes 2, 4, and 6) were incubated with the indicated substrate proteins. The substrat…
-
Figure 4—source data 1
Raw values used for the quantification of Figure 4C.
- https://doi.org/10.7554/eLife.33309.014
Figure 4—figure supplement 1
CDK–phosphorylation of ORC intrinsically inhibits helicase loading.
(A) CDK–phosphorylation of each protein separately shows that the primary target of CDK–mediated inhibition is ORC (compare lane 5 and 6). Top: Flowchart of experiment. Bottom: Helicase–loading …
Figure 5 with 2 supplements
CDK and Ime2 cooperate to prevent Mcm2–7 loading and inhibit CDC6 expression during the MI–MII transition.
Simultaneous inhibition of both CDK and Ime2 is required for robust Mcm2–7 reloading and CDC6 reaccumulation during the MI-MII transition. (A–D): Mcm2–7 loading (ChIP-qPCR), Orc2 phosphorylation …
-
Figure 5—source data 1
Raw values used for the quantification of Figure 5A–5D.
- https://doi.org/10.7554/eLife.33309.019
Figure 5—figure supplement 1
Cells in Figure 5 entered the MI-MII transition at the time of kinase inhibition.
(A–D) Cell–cycle stage quantification for Figure 5A–5D, respectively. At the time of treatment with 10 µM 1–NM–PP1 and 20 µM 1–NA–PP1, all strains had ~50% of cells in Anaphase I. The 8 hr 30’ …
Figure 5—figure supplement 2
CDK inhibition using a cdk1–as allele is sufficient for Mcm2–7 reloading in mitotic cells.
(A) Inhibition of CDK in G2/M arrested mitotic cells leads to helicase reloading. Cells from strain A4370 (cdk1-as) were arrested in alpha factor (G1) or nocodazole (G2/M). Nocodazole–arrested cells …
-
Figure 5—figure supplement 2—source data 1
Raw values used for the quantification of Figure 5—figure supplement 2.
- https://doi.org/10.7554/eLife.33309.018
Figure 6 with 3 supplements
Sld2 is degraded during the meiotic divisions in a manner that depends on Cdc5- and CDK-phosphorylation sites.
(A) Sld2 protein is degraded upon entry into the meiotic divisions. Immunoblots of Sld2–13myc during meiosis from strain yDP336. The time after transfer into sporulation medium and the associated …
-
Figure 6—source data 1
Raw values used for the quantification of Figure 6E.
- https://doi.org/10.7554/eLife.33309.025
Figure 6—figure supplement 1
Most helicase–activation proteins are present throughout the meiotic divisions.
(A–D) Top: Immunoblots for Cdc45–13myc, Sld3–13myc, Dpb11–3V5, and Psf2–3V5, respectively. The time after transfer into sporulation medium and the associated meiotic stages are indicated above each …
Figure 6—figure supplement 2
Cells from Figure 6 proceeded synchronously through meiosis.
(A–D) Cell–cycle stage quantification for Figure 6A–D, respectively.
Figure 6—figure supplement 3
Sld2-13myc and Pgk1 levels are quantifiable across a 32-fold dilution range.
(A) Sld2 and Pgk1 western blots have increased intensity with increased input. (B) Quantification of Sld2 and Pgk1 levels is similar to the expected value based on the relative amount of input.
-
Figure 6—figure supplement 3—source data 1
Raw values used for the quantification of Figure 6—figure supplement 3.
- https://doi.org/10.7554/eLife.33309.024
Figure 7
Model of how meiotic cells inhibit DNA replication during the MI–MII transition.
(A) Graphical representation of CDK (blue) (Carlile and Amon, 2008) and Ime2 (red) (Berchowitz et al., 2013) kinase activities during meiosis, and how they regulate the chromosome segregation and …
Author response image 1
OCCM Formation Assay.
https://doi.org/10.7554/eLife.33309.030Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background (Saccharomyces cerevisiae SK1) | yDP71 | This paper | Cdc6-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 CDC6-3V5::KANMX6/CDC6-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP120 | This paper | Orc6-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 ORC6-3V5::KANMX6/ORC6-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP152 | This paper | cdk1-as, Cdc6-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 cdc28-as1(F88G)/cdc28-as1(F88G) CDC6-3V5::KANMX6/CDC6-3V5::KANMX6 |
| strain, strain background (S. cerevisiae W303) | yDP159 | This paper | Ime2 Purification | W303 MATa bar1::hisG pep4::unmarked LEU2::pGAL1,10-IME2(1–404)−3xFLAG |
| strain, strain background (S. cerevisiae SK1) | yDP176 | This paper | ime2-as, Cdc6-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 ime2-as1(M146G)/ime2-as1(M146G) CDC6-3V5::KANMX6/CDC6-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP177 | This paper | cdk1-as, ime2-as, Cdc6-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 cdc28-as1(F88G)/cdc28-as1(F88G) ime2-as1(M146G)/ime2-as1(M146G) CDC6-3V5::KANMX6/CDC6-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP329 | This paper | Dpb11-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 DPB11-3V5::KANMX6/DPB11-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP330 | This paper | Psf2-3V5 | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 PSF2-3V5::KANMX6/PSF2-3V5::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP335 | This paper | Cdc45-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 CDC45-13myc::KANMX6/CDC45-13myc::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP336 | This paper | Sld2-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 SLD2-13myc::KANMX6/SLD2-13myc::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP337 | This paper | Sld3-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 SLD3-13myc::KANMX6/SLD3-13myc::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP473 | This paper | Sld2-2TA-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 SLD2(T122A T143A)−13myc::KANMX6/ SLD2(T122A T143A)−13myc::KANMX6 |
| strain, strain background (S. cerevisiae W303) | yDP554 | This paper | Ime2-AS Purification | W303 MATa bar1::hisG pep4::unmarked LEU2::pGAL1,10-IME2(1–404, M146G)−3xFLAG |
| strain, strain background (S. cerevisiae SK1) | yDP642 | This paper | Sld2-2SA-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 SLD2(S128A S138A)−13myc::KANMX6/ SLD2(S128A S138A)−13myc::KANMX6 |
| strain, strain background (S. cerevisiae SK1) | yDP644 | This paper | Sld2-4A-13myc | SK1 MATa/alpha ura3::pGPD1-GAL4(848). ER::URA3/ura3::pGPD1- GAL4(848).ER::URA3 GAL-NDT80::TRP1/GAL-NDT80::TRP1 SLD2(T122A S128A S138A T143A)−13myc:: KANMX6/SLD2(T122A S128A S138A T143A) −13myc::KANMX6 |
| strain, strain background (S. cerevisiae W303) | ySK119 | Lõoke et al, 2017 (PMID: 28270517) | Cdk1-Clb5 Purification | W303 MATa bar1::hisG pep4::unmarked URA3::pGAL1,10-Cdc28-His,Δ1–95-Clb5-Flag |
| strain, strain background (S. cerevisiae SK1) | yST135 | This paper | Mcm2-7 Purification | W303 MATa bar1::hisG pep4::unmarked TRP1::pSKM003(pGAL1,10-MCM6,MCM7) HIS3::pSKM004-(pGAL1,10-MCM2,Flag-MCM3) LYS2::pSKM002-(pGAL1,10-MCM4,MCM5) |
| strain, strain background (S. cerevisiae W303) | yST144 | Ticau et al. (2015) (PMID: 25892223) | Mcm2-7-Cdt1 Purification | W303 MATa bar1::hisG pep4::unmarked TRP1::pSKM003(pGAL1,10-MCM6,MCM7) HIS3::pSKM004-(pGAL1,10-MCM2,Flag-MCM3) LYS2::pSKM002-(pGAL1,10-MCM4,MCM5) URA3::pALS1(pGAL1,10-Cdt1,GAL4) |
| strain, strain background (S. cerevisiae W303) | A4370 | Angelika Amon | cdk1-as | W303 MATa bar1::hisG cdc28-as1(F88G) |
| strain, strain background (S. cerevisiae W303) | ySDORC | John Diffley | ORC purification | W303 MATa bar1::hyg pep4::kanMX TRP1::pGAL1,10-ORC5,ORC6 HIS3::pGAL1,10-ORC3,ORC4 URA3::pGAL1, 10-CBP-TEV-ORC1,ORC2 |
| antibody | poly ORC (Orc1 and Orc2 western blots and ORC ChIP) | HM1108 (Bell Lab) | ||
| antibody | Cdt1 | HM5353 (Bell Lab) | ||
| antibody | poly MCM (Mcm3 and Mcm6 western blots) | UM174 (Bell Lab) | ||
| antibody | poly MCM (Mcm2-7 ChIP) | UM185 (Bell Lab) | ||
| antibody | Mcm2 | Santa Cruz, yN-19 (code sc-6680) | RRID:AB_648843 | |
| antibody | Mcm7 | Santa Cruz, yN-19 (code SC-6688) | RRID:AB_647936 | |
| antibody | PGK1 | Invitrogen (catalog #459250) | RRID:AB_2532235 | |
| recombinant DNA reagent (plasmid) | pSKM033 | Kang et al. (2014) (PMID: 25087876) | Cdc6 purification | pGEX-GST-3C-FLAG-CDC6 |
| recombinant DNA reagent (plasmid) | pALS16 | This study | Cdt1 purification | pGEX-GST-3C-CDT1 |
| chemical compound, drug | 1-NM-PP1 | Toronto Research Chemicals (catalog #A603003) | ||
| chemical compound, drug | 1-NA-PP1 | Cayman Chemical Co., (catalog #NC1049860) |
Additional files
-
Supplementary file 1
In vitro Ime2–phosphorylation sites on Cdc6 from iTRAQ LC–MS/MS.
Related to Figure 5E. Shown are the phosphorylated Cdc6 peptides, the specific phosphorylated residue(s), the relative amount of those phosphopeptides detected in both biological replicates of buffer–treated and Ime2–treated Cdc6, and the average enrichment upon Ime2–treatment. See Materials and Methods for iTRAQ LC–MS/MS details.
- https://doi.org/10.7554/eLife.33309.027
-
Transparent reporting form
- https://doi.org/10.7554/eLife.33309.028
