DNA replication in eukaryotic cells initiates from large numbers of sites called replication origins. Initiation of replication from these origins must be tightly controlled to ensure the entire ...genome is precisely duplicated in each cell cycle. This is accomplished through the regulation of the first two steps in replication: loading and activation of the replicative DNA helicase. Here we describe what is known about the mechanism and regulation of these two reactions from a genetic, biochemical, and structural perspective, focusing on recent progress using proteins from budding yeast.
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA
. The mechanism of MCM ...double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer
. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites
. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex ...replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.
DNA replication must be tightly regulated to ensure that the genome is accurately duplicated during each cell cycle. When these regulatory mechanisms fail, replicative stress and DNA damage ensue. ...Activated oncogenes promote replicative stress, inducing a DNA damage response (DDR) early in tumorigenesis. Senescence or apoptosis result, forming a barrier against tumour progression. This may provide a selective pressure for acquisition of mutations in the DDR pathway during tumorigenesis. Despite its potential importance in early cancer development, the precise nature of oncogene-induced replicative stress remains poorly understood. Here, we review our current understanding of replication initiation and its regulation, describe mechanisms by which activated oncogenes might interfere with these processes and discuss how replicative stress might contribute to the genomic instability seen in cancers.
Hills and Diffley review how oncogenes induce replication stress and speculate that this may be an important mechanism in the etiology of cancer.
The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases. CDK ...phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2-4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the 'intra-S checkpoint' is primarily due to the inhibition of origin firing.
Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast ...Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.
Bidirectional replication from eukaryotic DNA replication origins requires the loading of two ring-shaped minichromosome maintenance (MCM) helicases around DNA in opposite orientations. MCM loading ...is orchestrated by binding of the origin recognition complex (ORC) to DNA, but how ORC coordinates symmetrical MCM loading is unclear. We used natural budding yeast DNA replication origins and synthetic DNA sequences to show that efficient MCM loading requires binding of two ORC molecules to two ORC binding sites. The relative orientation of these sites, but not the distance between them, was found to be critical for MCM loading in vitro and origin function in vivo. We propose that quasi-symmetrical loading of individual MCM hexamers by ORC and directed MCM translocation into double hexamers acts as a unifying mechanism for the establishment of bidirectional replication in archaea and eukaryotes.
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.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2–7) into ...prereplicative complexes (pre-RCs). Mcm2–7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2–7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2–7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1•Mcm2–7 are loaded cooperatively and result in association of stable, head-to-head Mcm2–7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2–7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.
MCM: one ring to rule them all Deegan, Tom D; Diffley, John FX
Current opinion in structural biology,
April 2016, 2016-Apr, 2016-04-00, 20160401, Letnik:
37
Journal Article
Recenzirano
•The eukaryotic MCM replicative helicase is tightly regulated during the cell cycle.•Conformational changes in MCM underpin the different stages of chromosome replication.•Reconstitution of DNA ...replication in vitro has revealed new features of MCM mechanism.
Precise replication of the eukaryotic genome is achieved primarily through strict regulation of the enzyme responsible for DNA unwinding, the replicative helicase. The motor of this helicase is a hexameric AAA+ ATPase called MCM. The loading of MCM onto DNA and its subsequent activation and disassembly are each restricted to separate cell cycle phases; this ensures that a functional replisome is only built once at any replication origin. In recent years, biochemical and structural studies have shown that distinct conformational changes in MCM, each requiring post-translational modifications and/or the activity of other replication proteins, define the various stages of the chromosome replication cycle. Here, we review recent progress in this area.
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