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It has long been a goal to reconstitute eukaryotic DNA replication; here a purified in vitro system from budding yeast containing 16 factors, themselves composed of 42 polypeptides, fulfils the staged process of origin-dependent initiation, including its regulation by kinases.

The authors describe how the eukaryotic replicative helicase is recruited to origins and reveal a novel ATPase-dependent quality control mechanism.

Licensing of eukaryotic origins of replication with MCM double hexamers (DHs) can occur through distinct pathways. Here, Lim et al. show that in yeast, cell cycle-dependent regulation of DH formation by CDK and origin structure have co-evolved.

Biochemical and structural studies of the human MCM replicative DNA helicase complex show differences from the yeast complex and provide insights into mechanisms of double hexamer assembly on sequence-independent replication origins.

In vitro experiments, using purified proteins and an assay that detects DNA unwinding, reveal the mechanism of activation of eukaryotic DNA replication.

Two classes of enzyme — cyclin-dependent kinases (CDK) and Dbf4-dependent kinase (DDK) — facilitate the initiation of DNA replication in eukaryotes. It is now shown that, when DNA damage is sensed, another kinase, Rad53, halts the firing of late replication origins by inhibiting both the CDK and the DDK pathways. Rad53 acts on DDK directly by inhibiting Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate Sld3.

This paper, one of two published that show Sld2 and Sld3 constitute the minimal group of cyclin-dependent kinase (CDK) targets necessary to promote replication, also finds that the function of G1 CDKs in replication is not just to activate the S-phase CDKs, but also to regulate the Cdc7 kinase regulatory subunit, Dbf4.

The loading and activation of the Mcm2-7 replicative helicase couples cell cycle progression to DNA replication. Here the authors use X-ray crystallography and single-particle electron microscopy to demonstrate how Ctd1 functions to promote MCM loading onto DNA.

Eukaryotic DNA replication is regulated to ensure copying of the genome (only) once per cell cycle. Here the authors, using optical trapping and confocal microscopy, demonstrate the dynamics of the origin recognition complex and subsequent intermediates that lead up to the loading of an MCM helicase onto DNA.

Here the authors examine fully reconstituted and activated CMG helicases at the single-molecule level. They observe unidirectional translocation by CMG in ATP and diffusive motion by CMG without ATP, where the latter can be halted by nucleotide binding.

Cryo-electron microscopy structures of the sequential assembly of the CMG replicative helicase on a chromatinized origin of replication provide insights into the mechanism through which DNA melting is initiated by ATP binding.

What prevents the replisome complex from moving along the DNA or breaking down when the replication fork stalls or encounters a DNA lesion? A recent paper describes how checkpoint proteins, Mrc1 and Tof1 couple the replisome to DNA synthesis.

Time-resolved electron microscopy reveals the mechanism by which the origin recognition complex loads pairs of MCM helicases around DNA prior to bidirectional replication.

Here the authors used cryogenic electron microscopy and biochemistry to understand how yeast Mcm10 exerts its essential role in DNA replication initiation, finding that it splits the double Cdc45-MCM-GINS-Polε structure. The lagging-strand template is ejected from each MCM ring as the central channel of the helicase becomes too tight to accommodate two DNA strands.

Origins of replication are licensed by loading of MCM onto DNA, and origin firing depends on interaction with Cdc45 and GINS to form two CMG holo-helicases. Here, authors determine the cryo-EM structures of DNA-bound MCM and visualise a phospho-dependent MCM element important for Cdc45 recruitment.

Here, the authors comprehensively discuss the different mechanisms for origin licensing in yeast and human cells. Importantly, they present intuitive videos illustrating such mechanisms with unprecedented detail.

A cryo-EM structure of the Dbf4-dependent kinase (DDK) in the act of phosphorylating an Mcm2-7 helicase complex reveals how DDK selectively targets DNA-loaded MCM double hexamers to trigger replication origin activation. Checkpoint kinase Rad53 blocks origin firing by impairing double hexamer engagement by DDK.

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase, which requires the leading-strand polymerase Pol ɛ. Here the authors present a structural analysis of a CMG Pol ɛ on a DNA fork, providing insight on the steps leading productive helicase engagement to the DNA junction.

Here the authors investigate the impact of chromatinizing origins of replication on ORC and MCM is at the single-molecule level. They find mobility of ORC reduced, but not its binding to the origin. MCM is both efficiently recruited and spatially confined to the origin.