Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6.

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA.

From structure to mechanism-understanding initiation of DNA replication.

DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin.

Hexokinase 2 Is an Intracellular Glucose Sensor of Yeast Cells That Maintains the Structure and Activity of Mig1 Protein Repressor Complex.

Hexokinase 2 (Hxk2) fromSaccharomyces cerevisiaeis a bi-functional enzyme, being both a catalyst in the cytosol and an important regulator of the glucose repression signal in the nucleus. Despite considerable recent progress, little is known about the regulatory mechanism that controls nuclear Hxk2 association with theSUC2promoter chromatin and how this association is necessary forSUC2gene repression. Our data indicate that in theSUC2promoter context, Hxk2 functions through a variety of structurally unrelated factors, mainly the DNA-binding Mig1 and Mig2 repressors and the regulatory Snf1 and Reg1 factors.

A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase.

During G1-phase of the cell-cycle the replicative MCM2-7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2-7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45-MCM2-7-GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2-7 double-hexamer represents a very large interaction surface.

Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication.

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading.

Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function.

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood.

Switch on the engine: how the eukaryotic replicative helicase MCM2-7 becomes activated.

A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2-7 (MCM2-7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle.

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading.

Helicase loading: how to build a MCM2-7 double-hexamer.

A central step in eukaryotic initiation of DNA replication is the loading of the helicase at replication origins, misregulation of this reaction leads to DNA damage and genome instability. Here we discuss how the helicase becomes recruited to origins and loaded into a double-hexamer around double-stranded DNA. We specifically describe the individual steps in complex assembly and explain how this process is regulated to maintain genome stability.

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation.

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex.

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer.

An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly.

In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6.

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization.

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation.

Glucose levels regulate the nucleo-mitochondrial distribution of Mig2.

Mig2 has been described as a transcriptional factor that in the absence of Mig1 protein is required for glucose repression of the SUC2 gene. Thus, until now, the main role assigned to Mig2 has been the functional redundancy to Mig1. In this study, we report that Mig2 has a double subcellular localization.

Human pancreatic beta-cell glucokinase: subcellular localization and glucose repression signalling function in the yeast cell.

Human GK(beta) (pancreatic beta-cell glucokinase) is the main glucose-phosphorylating enzyme in pancreatic beta-cells. It shares several structural, catalytic and regulatory properties with Hxk2 (hexokinase 2) from Saccharomyces cerevisiae. In fact, it has been previously described that expression of GK(beta) in yeast could replace Hxk2 in the glucose signalling pathway of S.

Hxk2 regulates the phosphorylation state of Mig1 and therefore its nucleocytoplasmic distribution.

Mig1 and Hxk2 are two major mediators of glucose repression in Saccharomyces cerevisiae. However, the mechanism by which Hxk2 participates in the glucose repression signaling pathway is not completely understood. Recently, it has been demonstrated that Hxk2 interacts with Mig1 to generate a repressor complex located in the nucleus of S.

A glucose response element from the S. cerevisiae hexose transporter HXT1 gene is sensitive to glucose in human fibroblasts.

Glucose is an essential nutrient, and a regulator of gene expression in eukaryotic cells. Here, a comparative, function-based genomic approach has been used to identify glucose regulatory elements and transduction pathways common to both yeast and mammalian cells. We have isolated a region in the promoter of the Saccharomyces cerevisiae hexose transporter gene HXT1 that conferred glucose sensitivity in yeast, when located upstream of the minimal CYC1 promoter.