Regulated eukaryotic DNA replication origin firing with purified proteins.
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.
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
Joseph T P
JT
Yeeles
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
Tom D
TD
Deegan
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
Agnieszka
A
Janska
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
Anne
A
Early
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
John F X
JF
Diffley
D018797:Cell Cycle Proteins / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics / Q000502:physiology*; D051716:Replication Protein A / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D000099272:Replisomes
D018797:Cell Cycle Proteins; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D064110:Minichromosome Maintenance Proteins; D009097:Multienzyme Complexes; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D000099272:Replisomes; C102209:CDC45 protein, S cerevisiae; C082728:RFA1 protein, S cerevisiae; C489404:CDC7 protein, S cerevisiae