Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins.

ABSTRACT: Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins.

SUBMITTER: Samson RY

PROVIDER: S-EPMC4724246 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins.

Samson Rachel Y RY Abeyrathne Priyanka D PD Bell Stephen D SD

Molecular cell 20151224 2

Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaea ...[more]

PMID: 26725007

Similar Datasets

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication.

Project description:Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin-modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome-reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin-modifying activities promotes chromosome replication.

| S-EPMC1560368 | biostudies-literature

Insights into the MCM functional mechanism: lessons learned from the archaeal MCM complex.

Project description:The helicase function of the minichromosome maintenance protein (MCM) is essential for genomic DNA replication in archaea and eukaryotes. There has been rapid progress in studies of the structure and function of MCM proteins from different organisms, leading to better understanding of the MCM helicase mechanism. Because there are a number of excellent reviews on this topic, we will use this review to summarize some of the recent progress, with particular focus on the structural aspects of MCM and their implications for helicase function. Given the hexameric and double hexameric architecture observed by X-ray crystallography and electron microscopy of MCMs from archaeal and eukaryotic cells, we summarize and discuss possible unwinding modes by either a hexameric or a double hexameric helicase. Additionally, our recent crystal structure of a full length archaeal MCM has provided structural information on an intact, multi-domain MCM protein, which includes the salient features of four unusual beta-hairpins from each monomer, and the side channels of a hexamer/double hexamer. These new structural data enable a closer examination of the structural basis of the unwinding mechanisms by MCM.

| S-EPMC2953368 | biostudies-literature

DNA translocation mechanism of the MCM complex and implications for replication initiation.

Project description:The DNA translocation activity of the minichromosome maintenance (MCM) complex powers DNA strand separation of the replication forks of eukaryotes and archaea. Here we illustrate an atomic level mechanism for this activity with a crystal structure of an archaeal MCM hexamer bound to single-stranded DNA and nucleotide cofactors. Sequence conservation indicates this rotary mechanism is fully possible for all eukaryotes and archaea. The structure definitively demonstrates the ring orients during translocation with the N-terminal domain leading, indicating that the translocation activity could also provide the physical basis of replication initiation where a double-hexamer idly encircling double-stranded DNA transforms to single-hexamers that encircle only one strand. In this mechanism, each strand binds to the N-terminal tier of one hexamer and the AAA+ tier of the other hexamer such that one ring pulls on the other, aligning equivalent interfaces to enable each hexamer to pull its translocation strand outside of the opposing hexamer.

| S-EPMC6629641 | biostudies-literature

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM.

Project description:By dissecting and reconstituting a cell-free influenza virus genome replication system, we have purified and identified the minichromosome maintenance (MCM) complex, which is thought to be a DNA replicative helicase, as one of the host factors that regulate the virus genome replication. MCM interacted with the PA subunit of the viral RNA-dependent RNA polymerase that is found to be involved in the replication genetically. The virus genome replication was decreased in MCM2 knockdown cells. The viral polymerase appeared to be a nonproductive complex, that is, it was capable of initiating replication but produced only abortive short RNA chains. MCM stimulated de novo-initiated replication reaction by stabilizing a replication complex during its transition from initiation to elongation. Based on the findings, including the result that the MCM-mediated RNA replication reaction was competed with exogenously added RNA, we propose that MCM functions as a scaffold between the nascent RNA chains and the viral polymerase.

| S-EPMC2063485 | biostudies-literature

Archaeal DNA Replication.

Project description:It is now well recognized that the information processing machineries of archaea are far more closely related to those of eukaryotes than to those of their prokaryotic cousins, the bacteria. Extensive studies have been performed on the structure and function of the archaeal DNA replication origins, the proteins that define them, and the macromolecular assemblies that drive DNA unwinding and nascent strand synthesis. The results from various archaeal organisms across the archaeal domain of life show surprising levels of diversity at many levels-ranging from cell cycle organization to chromosome ploidy to replication mode and nature of the replicative polymerases. In the following, we describe recent advances in the field, highlighting conserved features and lineage-specific innovations.

| S-EPMC7712474 | biostudies-literature

The Mcm complex: unwinding the mechanism of a replicative helicase.

Project description:The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.

| S-EPMC2786579 | biostudies-literature

The capacity of origins to load MCM establishes replication timing patterns.

Project description:Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins' ability to recruit ORC and compete for MCM when MCM becomes limiting.

| S-EPMC8023499 | biostudies-literature

The Helicase Activity of Hyperthermophilic Archaeal MCM is Enhanced at High Temperatures by Lysine Methylation.

Project description:Lysine methylation and methyltransferases are widespread in the third domain of life, archaea. Nevertheless, the effects of methylation on archaeal proteins wait to be defined. Here, we report that recombinant sisMCM, an archaeal homolog of Mcm2-7 eukaryotic replicative helicase, is methylated by aKMT4 in vitro. Mono-methylation of these lysine residues occurs coincidently in the endogenous sisMCM protein purified from the hyperthermophilic Sulfolobus islandicus cells as indicated by mass spectra. The helicase activity of mini-chromosome maintenance (MCM) is stimulated by methylation, particularly at temperatures over 70°C. The methylated MCM shows optimal DNA unwinding activity after heat-treatment between 76 and 82°C, which correlates well with the typical growth temperatures of hyperthermophilic Sulfolobus. After methylation, the half life of MCM helicase is dramatically extended at 80°C. The methylated sites are located on the accessible protein surface, which might modulate the intra- and inter- molecular interactions through changing the hydrophobicity and surface charge. Furthermore, the methylation-mimic mutants of MCM show heat resistance helicase activity comparable to the methylated MCM. These data provide the biochemical evidence that posttranslational modifications such as methylation may enhance kinetic stability of proteins under the elevated growth temperatures of hyperthermophilic archaea.

| S-EPMC4639711 | biostudies-literature

Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks.

Project description:The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2-7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.

| S-EPMC2529087 | biostudies-literature

Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase.

Project description:The minichromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. Whereas the eukaryotic complex consists of 6 homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only 1 MCM protein (ssoMCM), 6 subunits of which form a homohexamer. Here, we report a 4.35-A crystal structure of the near-full-length ssoMCM. The structure shows an elongated fold, with 5 subdomains that are organized into 2 large N- and C-terminal domains. A near-full-length ssoMCM hexamer generated based on the 6-fold symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) hexamer shows intersubunit distances suitable for bonding contacts, including the interface around the ATP pocket. Four unusual beta-hairpins of each subunit are located inside the central channel or around the side channels in the hexamer. Additionally, the hexamer fits well into the double-hexamer EM map of mtMCM. Our mutational analysis of residues at the intersubunit interfaces and around the side channels demonstrates their critical roles for hexamerization and helicase function. These structural and biochemical results provide a basis for future study of the helicase mechanisms of the archaeal and eukaryotic MCM complexes in DNA replication.

| S-EPMC2629282 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data