Regulated eukaryotic DNA replication origin firing with purified proteins

Joseph T P Yeeles; Tom D Deegan; Agnieszka Janska; Anne Early; John F X Diffley

doi:10.1038/nature14285

Regulated eukaryotic DNA replication origin firing with purified proteins

Joseph T P Yeeles, Tom D Deegan, Agnieszka Janska, Anne Early, John F X Diffley

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Original language	English
Pages (from-to)	431-5
Number of pages	5
Journal	Nature
Volume	519
Issue number	7544
DOIs	https://doi.org/10.1038/nature14285
Publication status	Published - 26 Mar 2015

Keywords / Materials (for Non-textual outputs)

Cell Cycle Proteins/metabolism
Cyclin-Dependent Kinases/metabolism
DNA Replication
DNA-Binding Proteins/metabolism
DNA-Directed DNA Polymerase/metabolism
Minichromosome Maintenance Proteins/metabolism
Multienzyme Complexes/metabolism
Multiprotein Complexes/chemistry
Nuclear Proteins/metabolism
Phosphorylation
Protein-Serine-Threonine Kinases/metabolism
Replication Origin/genetics
Replication Protein A/metabolism
Saccharomyces cerevisiae/enzymology
Saccharomyces cerevisiae Proteins/isolation & purification

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10.1038/nature14285

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@article{2ca3b52c5fb04cf39510f61ee7063859,

title = "Regulated eukaryotic DNA replication origin firing with purified proteins",

abstract = "Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. ",

keywords = "Cell Cycle Proteins/metabolism, Cyclin-Dependent Kinases/metabolism, DNA Replication, DNA-Binding Proteins/metabolism, DNA-Directed DNA Polymerase/metabolism, Minichromosome Maintenance Proteins/metabolism, Multienzyme Complexes/metabolism, Multiprotein Complexes/chemistry, Nuclear Proteins/metabolism, Phosphorylation, Protein-Serine-Threonine Kinases/metabolism, Replication Origin/genetics, Replication Protein A/metabolism, Saccharomyces cerevisiae/enzymology, Saccharomyces cerevisiae Proteins/isolation & purification",

author = "Yeeles, {Joseph T P} and Deegan, {Tom D} and Agnieszka Janska and Anne Early and Diffley, {John F X}",

year = "2015",

month = mar,

day = "26",

doi = "10.1038/nature14285",

language = "English",

volume = "519",

pages = "431--5",

journal = "Nature",

issn = "0028-0836",

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T1 - Regulated eukaryotic DNA replication origin firing with purified proteins

AU - Yeeles, Joseph T P

AU - Deegan, Tom D

AU - Janska, Agnieszka

AU - Early, Anne

AU - Diffley, John F X

PY - 2015/3/26

Y1 - 2015/3/26

N2 - Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

AB - Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

KW - Cell Cycle Proteins/metabolism

KW - Cyclin-Dependent Kinases/metabolism

KW - DNA Replication

KW - DNA-Binding Proteins/metabolism

KW - DNA-Directed DNA Polymerase/metabolism

KW - Minichromosome Maintenance Proteins/metabolism

KW - Multienzyme Complexes/metabolism

KW - Multiprotein Complexes/chemistry

KW - Nuclear Proteins/metabolism

KW - Phosphorylation

KW - Protein-Serine-Threonine Kinases/metabolism

KW - Replication Origin/genetics

KW - Replication Protein A/metabolism

KW - Saccharomyces cerevisiae/enzymology

KW - Saccharomyces cerevisiae Proteins/isolation & purification

U2 - 10.1038/nature14285

DO - 10.1038/nature14285

M3 - Article

C2 - 25739503

SN - 0028-0836

VL - 519

SP - 431

EP - 435

JO - Nature

JF - Nature

IS - 7544

ER -

Regulated eukaryotic DNA replication origin firing with purified proteins

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

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