ABSTRACT Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation,

which triggers ubiquitination by SCFCdc4. We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7–Dbf4 and the GSK-3 homolog Mck1. These

kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced

correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing

ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print issues and online access $209.00 per year only $17.42 per issue Learn more Buy this article *

Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn

about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS ATM SIGNALING MODULATES COHESIN BEHAVIOR IN MEIOTIC PROPHASE AND

PROLIFERATING CELLS Article Open access 06 March 2023 CONFORMATIONAL DYNAMICS OF COHESIN/SCC2 LOADING COMPLEX ARE REGULATED BY SMC3 ACETYLATION AND ATP BINDING Article Open access 22

September 2023 COORDINATION OF COHESIN AND DNA REPLICATION OBSERVED WITH PURIFIED PROTEINS Article 24 January 2024 REFERENCES * Holt, L.J. et al. Global analysis of Cdk1 substrate

phosphorylation sites provides insights into evolution. _Science_ 325, 1682–1686 (2009). Article  CAS  Google Scholar  * Ubersax, J.A. et al. Targets of the cyclin-dependent kinase Cdk1.

_Nature_ 425, 859–864 (2003). Article  CAS  Google Scholar  * Petroski, M.D. & Deshaies, R.J. Function and regulation of cullin-RING ubiquitin ligases. _Nat. Rev. Mol. Cell Biol._ 6,

9–20 (2005). Article  CAS  Google Scholar  * Welcker, M. & Clurman, B.E. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. _Nat.

Rev. Cancer_ 8, 83–93 (2008). Article  CAS  Google Scholar  * Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. _Nature_ 414,

514–521 (2001). Article  CAS  Google Scholar  * Hao, B., Oehlmann, S., Sowa, M.E., Harper, J.W. & Pavletich, N.P. Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated

Substrate Recognition by SCF Ubiquitin Ligases. _Mol. Cell_ 26, 131–143 (2007). Article  CAS  Google Scholar  * Bao, M.Z., Shock, T.R. & Madhani, H.D. Multisite phosphorylation of the

_Saccharomyces cerevisiae_ filamentous growth regulator Tec1 is required for its recognition by the E3 ubiquitin ligase adaptor Cdc4 and its subsequent destruction _in vivo_. _Eukaryot.

Cell_ 9, 31–36 (2010). Article  CAS  Google Scholar  * Welcker, M. & Clurman, B.E. Fbw7/hCDC4 dimerization regulates its substrate interactions. _Cell Div._ 2, 7 (2007). Article  Google

Scholar  * Tang, X. et al. Suprafacial orientation of the SCFCdc4 dimer accommodates multiple geometries for substrate ubiquitination. _Cell_ 129, 1165–1176 (2007). Article  CAS  Google

Scholar  * Lyons, N.A. & Morgan, D.O. Cdk1-dependent destruction of Eco1 prevents cohesion establishment after S phase. _Mol. Cell_ 42, 378–389 (2011). Article  CAS  Google Scholar  *

Rolef Ben-Shahar, T. et al. Eco1-dependent cohesin acetylation during establishment of sister chromatid cohesion. _Science_ 321, 563–566 (2008). Article  Google Scholar  * Rowland, B.D. et

al. Building sister chromatid cohesion: smc3 acetylation counteracts an antiestablishment activity. _Mol. Cell_ 33, 763–774 (2009). Article  CAS  Google Scholar  * Sutani, T., Kawaguchi, T.,

Kanno, R., Itoh, T. & Shirahige, K. Budding yeast Wpl1(Rad61)-Pds5 complex counteracts sister chromatid cohesion-establishing reaction. _Curr. Biol._ 19, 492–497 (2009). Article  CAS 

Google Scholar  * Unal, E. et al. A molecular determinant for the establishment of sister chromatid cohesion. _Science_ 321, 566–569 (2008). Article  Google Scholar  * Ström, L. et al.

Postreplicative formation of cohesion is required for repair and induced by a single DNA break. _Science_ 317, 242–245 (2007). Article  Google Scholar  * Ström, L., Lindroos, H.B.,

Shirahige, K. & Sjogren, C. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. _Mol. Cell_ 16, 1003–1015 (2004). Article  Google Scholar  * Unal,

E., Heidinger-Pauli, J.M. & Koshland, D. DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7). _Science_ 317, 245–248 (2007). Article  Google

Scholar  * Sjögren, C. & Nasmyth, K. Sister chromatid cohesion is required for postreplicative double-strand break repair in _Saccharomyces cerevisiae_. _Curr. Biol._ 11, 991–995 (2001).

Article  Google Scholar  * Heidinger-Pauli, J.M., Unal, E., Guacci, V. & Koshland, D. The kleisin subunit of cohesin dictates damage-induced cohesion. _Mol. Cell_ 31, 47–56 (2008).

Article  CAS  Google Scholar  * Heidinger-Pauli, J.M., Unal, E. & Koshland, D. Distinct targets of the Eco1 acetyltransferase modulate cohesion in S phase and in response to DNA damage.

_Mol. Cell_ 34, 311–321 (2009). Article  CAS  Google Scholar  * Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. & Koike, T. Phosphate-binding tag, a new tool to visualize

phosphorylated proteins. _Mol. Cell. Proteomics_ 5, 749–757 (2006). Article  CAS  Google Scholar  * Cho, W.H., Lee, Y.J., Kong, S.I., Hurwitz, J. & Lee, J.K. CDC7 kinase phosphorylates

serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein. _Proc. Natl. Acad. Sci. USA_ 103, 11521–11526 (2006). Article  CAS  Google Scholar  * Masai, H. et

al. Human Cdc7-related kinase complex. _In vitro_ phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a criticial threonine residue of Cdc7 bY Cdks. _J. Biol. Chem._

275, 29042–29052 (2000). Article  CAS  Google Scholar  * Mok, J. et al. Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs. _Sci.

Signal._ 3, ra12 (2010). Article  Google Scholar  * Randell, J.C. et al. Mec1 is one of multiple kinases that prime the Mcm2–7 helicase for phosphorylation by Cdc7. _Mol. Cell_ 40, 353–363

(2010). Article  CAS  Google Scholar  * Oshiro, G., Owens, J.C., Shellman, Y., Sclafani, R.A. & Li, J.J. Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p

stability. _Mol. Cell. Biol._ 19, 4888–4896 (1999). Article  CAS  Google Scholar  * Sullivan, M., Holt, L. & Morgan, D.O. Cyclin-specific control of ribosomal DNA segregation. _Mol.

Cell. Biol._ 28, 5328–5336 (2008). Article  CAS  Google Scholar  * Labib, K. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?

_Genes Dev._ 24, 1208–1219 (2010). Article  CAS  Google Scholar  * Marston, A.L. Meiosis: DDK is not just for replication. _Curr. Biol._ 19, R74–R76 (2009). Article  CAS  Google Scholar  *

Jackson, A.L., Pahl, P.M., Harrison, K., Rosamond, J. & Sclafani, R.A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein. _Mol. Cell. Biol._ 13,

2899–2908 (1993). Article  CAS  Google Scholar  * Doble, B.W. & Woodgett, J.R. GSK-3: tricks of the trade for a multi-tasking kinase. _J. Cell Sci._ 116, 1175–1186 (2003). Article  CAS

  Google Scholar  * Fiol, C.J., Mahrenholz, A.M., Wang, Y., Roeske, R.W. & Roach, P.J. Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular

mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. _J. Biol. Chem._ 262, 14042–14048 (1987). CAS  Google Scholar  * Xu, C., Kim, N.G. & Gumbiner,

B.M. Regulation of protein stability by GSK3 mediated phosphorylation. _Cell Cycle_ 8, 4032–4039 (2009). Article  CAS  Google Scholar  * Kassir, Y., Rubin-Bejerano, I. &

Mandel-Gutfreund, Y. The _Saccharomyces cerevisiae_ GSK-3 beta homologs. _Curr. Drug Targets_ 7, 1455–1465 (2006). Article  CAS  Google Scholar  * Mizunuma, M., Hirata, D., Miyaoka, R. &

Miyakawa, T. GSK-3 kinase Mck1 and calcineurin coordinately mediate Hsl1 down-regulation by Ca2+ in budding yeast. _EMBO J._ 20, 1074–1085 (2001). Article  CAS  Google Scholar  * Kishi, T.,

Ikeda, A., Nagao, R. & Koyama, N. The SCFCdc4 ubiquitin ligase regulates calcineurin signaling through degradation of phosphorylated Rcn1, an inhibitor of calcineurin. _Proc. Natl.

Acad. Sci. USA_ 104, 17418–17423 (2007). Article  CAS  Google Scholar  * Ikui, A.E., Rossio, V., Schroeder, L. & Yoshida, S. A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to

inhibit DNA re-replication. _PLoS Genet._ 8, e1003099 (2012). Article  CAS  Google Scholar  * Kõivomägi, M. et al. Cascades of multisite phosphorylation control Sic1 destruction at the onset

of S phase. _Nature_ 480, 128–131 (2011). Article  Google Scholar  * Tang, X. et al. Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by

the SCFCdc4 ubiquitin ligase. _Proc. Natl. Acad. Sci. USA_ 109, 3287–3292 (2012). Article  CAS  Google Scholar  * Liu, Q. et al. SCFCdc4 enables mating type switching in yeast by

cyclin-dependent kinase-mediated elimination of the Ash1 transcriptional repressor. _Mol. Cell. Biol._ 31, 584–598 (2011). Article  CAS  Google Scholar  * Kihara, M. et al. Characterization

of the yeast Cdc7p/Dbf4p complex purified from insect cells. Its protein kinase activity is regulated by Rad53p. _J. Biol. Chem._ 275, 35051–35062 (2000). Article  CAS  Google Scholar  *

Weinreich, M. & Stillman, B. Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. _EMBO J._ 18, 5334–5346 (1999).

Article  CAS  Google Scholar  * Lei, M. et al. Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. _Genes Dev._ 11, 3365–3374 (1997). Article  CAS  Google

Scholar  * Gabrielse, C. et al. A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast. _Genetics_ 173, 541–555 (2006). Article  CAS 

Google Scholar  * Takeda, T. et al. Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in

the fission yeast. _Mol. Biol. Cell_ 12, 1257–1274 (2001). Article  CAS  Google Scholar  * Lopez-Mosqueda, J. et al. Damage-induced phosphorylation of Sld3 is important to block late origin

firing. _Nature_ 467, 479–483 (2010). Article  CAS  Google Scholar  * Zegerman, P. & Diffley, J.F. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4

phosphorylation. _Nature_ 467, 474–478 (2010). Article  CAS  Google Scholar  * Mantiero, D., Mackenzie, A., Donaldson, A. & Zegerman, P. Limiting replication initiation factors execute

the temporal programme of origin firing in budding yeast. _EMBO J._ 30, 4805–4814 (2011). Article  CAS  Google Scholar  * Tanaka, S., Nakato, R., Katou, Y., Shirahige, K. & Araki, H.

Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. _Curr. Biol._ 21, 2055–2063 (2011). Article  CAS  Google Scholar  * Longtine,

M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in _Saccharomyces cerevisiae_. _Yeast_ 14, 953–961 (1998). Article  CAS  Google Scholar 

* Wolters, D.A., Washburn, M.P. & Yates, J.R. III. An automated multidimensional protein identification technology for shotgun proteomics. _Anal. Chem._ 73, 5683–5690 (2001). Article 

CAS  Google Scholar  * Fonslow, B.R. et al. Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with

MudPIT. _J. Proteome Res._ 11, 2697–2709 (2012). Article  CAS  Google Scholar  * Cociorva, D., Tabb, D.L. & Yates, J.R. Validation of tandem mass spectrometry database search results

using DTASelect. _Curr Protoc Bioinformatics_ CHAPTER 13, Unit 13 4 (2007). * Tabb, D.L., McDonald, W.H. & Yates, J.R. III. DTASelect and Contrast: tools for assembling and comparing

protein identifications from shotgun proteomics. _J. Proteome Res._ 1, 21–26 (2002). Article  CAS  Google Scholar  * Schroeder, M.J., Shabanowitz, J., Schwartz, J.C., Hunt, D.F. & Coon,

J.J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry. _Anal. Chem._ 76, 3590–3598 (2004). Article  CAS  Google Scholar

  * Breci, L.A., Tabb, D.L., Yates, J.R. III & Wysocki, V.H. Cleavage N-terminal to proline: analysis of a database of peptide tandem mass spectra. _Anal. Chem._ 75, 1963–1971 (2003).

Article  CAS  Google Scholar  * Courcelles, M., Bridon, G., Lemieux, S. & Thibault, P. Occurrence and detection of phosphopeptide isomers in large-scale phosphoproteomics experiments.

_J. Proteome Res._ 11, 3753–3765 (2012). Article  CAS  Google Scholar  * Loog, M. & Morgan, D.O. Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. _Nature_

434, 104–108 (2005). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank S. Bell (Massachusetts Institute of Technology, Cambridge, Massachusetts, USA), D. Toczyski

(University of California, San Francisco, California, USA), H. Madhani (University of California, San Francisco, California, USA) and K. Shokat (University of California, San Francisco,

California, USA) for strains and reagents, M. Loog for advice with kinase assays, A. Ikui for discussion of unpublished results, J. Wohlschlegel for advice with mass spectrometry, J.

Mugridge for assistance with fluorescence anisotropy and E. Edenberg and S. Foster for critical review of the manuscript. This work was supported by funding from the US National Institute of

General Medical Sciences (R01-GM069901 to D.O.M. and P41-GM103533 to J.R.Y.) and the National Center for Research Resources (P41-RR011823 to J.R.Y.). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Physiology, University of California, San Francisco, San Francisco, California, USA., Nicholas A Lyons & David O Morgan * Department of Chemical Physiology,

The Scripps Research Institute, La Jolla, California, USA., Bryan R Fonslow, Jolene K Diedrich & John R Yates III Authors * Nicholas A Lyons View author publications You can also search

for this author inPubMed Google Scholar * Bryan R Fonslow View author publications You can also search for this author inPubMed Google Scholar * Jolene K Diedrich View author publications

You can also search for this author inPubMed Google Scholar * John R Yates III View author publications You can also search for this author inPubMed Google Scholar * David O Morgan View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS N.A.L. and D.O.M. conceived the experiments, N.A.L. conducted the biological and biochemical

experiments, B.R.F. performed mass spectrometry, and B.R.F. and J.K.D. analyzed mass spectra under the guidance of J.R.Y. N.A.L. and D.O.M. wrote the manuscript. CORRESPONDING AUTHOR

Correspondence to David O Morgan. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES

Supplementary Figures 1 and 2 and Supplementary Tables 1 and 2 (PDF 449 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lyons, N., Fonslow, B.,

Diedrich, J. _et al._ Sequential primed kinases create a damage-responsive phosphodegron on Eco1. _Nat Struct Mol Biol_ 20, 194–201 (2013). https://doi.org/10.1038/nsmb.2478 Download

citation * Received: 31 July 2012 * Accepted: 27 November 2012 * Published: 13 January 2013 * Issue Date: February 2013 * DOI: https://doi.org/10.1038/nsmb.2478 SHARE THIS ARTICLE Anyone you

share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the

Springer Nature SharedIt content-sharing initiative