17 research outputs found
Structural and biochemical characterisation of DNA polymerase epsilon and its role in DNA replication
No full textDNA is copied by the replisome, a multi-protein assembly that couples polymerase and helicase activities. In eukaryotes, there are three main replicative polymerases (α, ε, δ) and the CMG (Cdc45-MCM-GINS) helicase is a three-member complex, where MCM (minichromosome maintenance) constitutes the motor while Cdc45 (cell division cycle 45) and GINS (go-ichi-ni-san) are essential cofactors. Pol ε is unique in that the protein is essential while cells where the catalytic domain is deleted become severely sick yet are still viable. Therefore, it was believed to have an additional role apart from DNA synthesis and proposed to recruit GINS to MCM together with Sld2 (synthetic lethal with Dpb11) and Dpb11 (DNA polymerase B-binding protein subunit 11) as part of the pre-LC (pre-loading complex). The role of Pol ε in helicase assembly was investigated by biochemical approaches and its structure was determined by electron microscopy both in isolation and in complex with CMG in order to investigate the helicase-polymerase coupling. The structural part of this work was performed in collaboration with Alessandro Costa’s Macromolecular Machines Laboratory and all microscopy work as well as data analysis was performed by the collaborators. The non-catalytic part of Pol ε was found to bind Sld2, Dpb11 and GINS while the catalytic domain was found to interact with Sld3. These results support the notion that the essential role of Pol ε is to aid CMG assembly. Although the pre-LC was reconstituted in vitro and independently of origins, it is still not clear whether its formation is strictly required. Pol ε was found to be made up of two lobes connected by a thin linker, which spatially separates the catalytic and non-catalytic portions of the assembly. The inactive lobe appears anchored at the front of the helicase while the catalytic lobe extends towards the side of CMG and adapts two conformations. Such positioning of the polymerase deviates from the classical model of the replisome, where polymerases trail behind the helicase and raises questions about the path of DNA through this assembly. The role of the conformational switch is not known, but it may be important for substrate engagement by Pol ε
Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading.
No full textBidirectional 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
Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro.
No full textTelomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process
DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p
No full textCdc45p assembles at replication origins before initiation and is required for origin firing in Saccharomyces cerevisiae. A heat-inducible cdc45 degron mutant was constructed that promotes rapid degradation of Cdc45p at the restrictive temperature, Consistent with a role in initiation, loss of Cdc45p in G(I) prevents all detectable DNA replication without preventing subsequent entry into mitosis, Loss of Cdc45p activity during S-phase blocks S-phase completion but not activation of replication checkpoints. Using density substitution, me show that after allowing replication fork establishment, Cdc45p inactivation prevents the subsequent progression of individual replication forks, This provides the first direct functional evidence that Cdc45p plays an essential role during elongation. Thus, like the large T antigen in SV40 replication, Cdc45p plays a central role in both initiation and elongation phases of chromosomal DNA replication.</p
Uninterrupted MCM2-7 function required for DNA replication fork progression
No full textLittle is known about the DNA helicases required for the elongation phase of eukaryotic chromosome replication. Minichromosone maintenance (MCM) protein complexes have DNA helicase activity but have only been functionally implicated in initiating DNA replication. Using an improved method for constructing conditional degron mutants, we show that depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae. Like the Escherichia coli dnaB and SV40 T antigen helicases, therefore, the MCM complex is loaded at origins before initiation and is essential for elongation. Restricting MCM Loading to the G(1) phase ensures that initiation and elongation occur just once per cell cycle.</p
Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.
No full textLoading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs
G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus
No full textCyclin-dependent kinases (CDKs) activate the firing of replication origins during the S phase of the cell cycle, They also block re-initiation of DNA replication within a single cell cycle, by preventing the assembly of prereplicative complexes at origins. We show here that, in budding yeast, CDKs exclude the essential prereplicative-complex component Mcm4 from the nucleus. Although origin firing can be triggered by the B-type cyclins only, both G1-phase and B-type cyclins cause exit of Mcm4 from the nucleus. These results suggest that G1 cyclins may diminish the cell's capacity to assemble prereplicative complexes before B-type cyclins trigger origin firing during S phase.</p
Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication.
No full textEukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins
Coping with and recovering from hydroxyurea-induced replication fork arrest in budding yeast
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