Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

By Joseph T. P. Yeeles, T. Deegan, Agnieszka Janska, A. Early, J. Diffley. 2015

516 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

• MCMBP promotes the assembly of the MCM2–7 hetero-hexamer to ensure robust DNA replication in human cells (2022, background): “Discussion Although a recombinant MCM2–7 hexamer has successfully been reconstructed using an overexpression system based on yeast, insect, and human cells (JenkynBedford et al., 2021; Rzechorzek et al., 2020; Yeeles et al., 2015), little is known about how the MCM2–7 hexamer is assembled in living ”
• DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7 (2020, methodology): “Elevating DDK concentration five-fold further (to 32.5 nM), increased the fraction of loaded Mcm2-7 complexes forming CMGs from 0.53 ± 0.04 to 0.69 ± 0.06 (Figure 3A).”