Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation

Tom D. Deegan, Joseph T P Yeeles, John F X Diffley (Lead / Corresponding author)

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

Original languageEnglish
JournalEMBO Journal
Volume35
Early online date24 Feb 2016
DOIs
Publication statusPublished - 2016

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Phosphopeptides
Multicarrier modulation
Telecommunication links
Phosphotransferases
Chemical activation
Cyclin-Dependent Kinases
DNA Replication
DNA
Replication Origin
Phosphorylation
S Phase
Protein Kinases
DNA Damage
Cell Cycle
Cells
Peptides
Substrates

Keywords

  • DDK
  • DNA replication initiation
  • Sld3

Cite this

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title = "Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation",
abstract = "The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential {"}reader{"} of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.",
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Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. / Deegan, Tom D.; Yeeles, Joseph T P; Diffley, John F X (Lead / Corresponding author).

In: EMBO Journal, Vol. 35, 2016.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation

AU - Deegan, Tom D.

AU - Yeeles, Joseph T P

AU - Diffley, John F X

PY - 2016

Y1 - 2016

N2 - The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

AB - The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

KW - DDK

KW - DNA replication initiation

KW - Sld3

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