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

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.

Fine-tuning of the replisome: Mcm10 regulates fork progression and regression.

Several decades of research have identified Mcm10 hanging around the replisome making several critical contacts with a number of proteins but with no real disclosed function. Recently, the O'Donnell laboratory has been better able to map the interactions of Mcm10 with a larger Cdc45/GINS/MCM (CMG) unwinding complex placing it at the front of the replication fork. They have shown biochemically that Mcm10 has the impressive ability to strip off single-strand binding protein (RPA) and reanneal complementary DNA strands. This has major implications in controlling DNA unwinding speed as well as responding to various situations where fork reversal is needed. This work opens up a number of additional facets discussed here revolving around accessing the DNA junction for different molecular purposes within a crowded replisome. Abbreviations: alt-NHEJ: Alternative Nonhomologous End-Joining; CC: Coli-Coil motif; CMG: Cdc45/GINS/MCM2-7; CMGM: Cdc45/GINS/Mcm2-7/Mcm10; CPT: Camptothecin; CSB: Cockayne Syndrome Group B protein; CTD: C-Terminal Domain; DSB: Double-Strand Break; DSBR: Double-Strand Break Repair; dsDNA: Double-Stranded DNA; GINS: go-ichi-ni-san, Sld5-Psf1-Psf2-Psf3; HJ Dis: Holliday Junction dissolution; HJ Res: Holliday Junction resolution; HR: Homologous Recombination; ICL: Interstrand Cross-Link; ID: Internal Domain; MCM: Minichromosomal Maintenance; ND: Not Determined; NTD: N-Terminal Domain; PCNA: Proliferating Cell Nuclear Antigen; RPA: Replication Protein A; SA: Strand Annealing; SE: Strand Exchange; SEW: Steric Exclusion and Wrapping; ssDNA: Single-Stranded DNA; TCR: Transcription-Coupled Repair; TOP1: Topoisomerase.

Nuclear Prohibitin3 Maintains Genome Integrity and Cell Proliferation in the Root Meristem through Minichromosome Maintenance 2.

The nucleo-mitochondrial dual-localized proteins can act as gene expression regulators; however, few instances of these proteins have been described in plants. Arabidopsis (Arabidopsis thaliana) PROHIBITIN 3 (PHB3) is involved in stress responses and developmental processes, but it is unknown how these roles are achieved at the molecular level in the nucleus. In this study, we show that nucleo-mitochondrial PHB3 plays an essential role in regulating genome stability and cell proliferation. PHB3 is up-regulated by DNA damage agents, and the stress-induced PHB3 proteins accumulate in the nucleus. Loss of function of PHB3 results in DNA damage and defective maintenance of the root stem cell niche. Subsequently, the expression patterns and levels of the root stem cell regulators are altered and down-regulated, respectively. In addition, the phb3 mutant shows aberrant cell division and altered expression of cell cycle-related genes, such as CycB1 and Cyclin dependent kinase 1 Moreover, the minichromosome maintenance (MCM) genes, e.g. MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, are up-regulated in the phb3 mutant. Reducing the MCM2 expression level substantially recovers the DNA damage in the phb3 mutant and partially rescues the altered cell proliferation and root deficiency of phb3 seedlings. PHB3 acts as a transcriptional coregulator that represses MCM2 expression by competitively binding to the promoter E2F-cis-acting elements with E2Fa so as to modulate primary root growth. Collectively, these findings indicate that nuclear-localized PHB3 acts as a transcriptional coregulator that suppresses MCM2 expression to sustain genome integrity and cell proliferation for stem cell niche maintenance in Arabidopsis.

Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication.

The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.

Replication origin-flanking roadblocks reveal origin-licensing dynamics and altered sequence dependence.

In eukaryotes, DNA replication initiates from multiple origins of replication for timely genome duplication. These sites are selected by origin licensing, during which the core enzyme of the eukaryotic DNA replicative helicase, the Mcm2-7 (minichromosome maintenance) complex, is loaded at each origin. This origin licensing requires loading two Mcm2-7 helicases around origin DNA in a head-to-head orientation. Current models suggest that the origin-recognition complex (ORC) and cell-division cycle 6 (Cdc6) proteins recognize and encircle origin DNA and assemble an Mcm2-7 double-hexamer around adjacent double-stranded DNA. To test this model and assess the location of Mcm2-7 initial loading, we placed DNA-protein roadblocks at defined positions adjacent to the essential ORC-binding site within Saccharomyces cerevisiae origin DNA. Roadblocks were made either by covalent cross-linking of the HpaII methyltransferase to DNA or through binding of a transcription activator-like effector (TALE) protein. Contrary to the sites of Mcm2-7 recruitment being precisely defined, only single roadblocks that inhibited ORC-DNA binding showed helicase loading defects. We observed inhibition of helicase loading without inhibition of ORC-DNA binding only when roadblocks were placed on both sides of the origin to restrict sliding of a helicase-loading intermediate. Consistent with a sliding helicase-loading intermediate, when either one of the flanking roadblocks was eliminated, the remaining roadblock had no effect on helicase loading. Interestingly, either origin-flanking nucleosomes or roadblocks resulted in helicase loading being dependent on an additional origin sequence known to be a weaker ORC-DNA-binding site. Together, our findings support a model in which sliding helicase-loading intermediates increase the flexibility of the DNA sequence requirements for origin licensing.

Overexpression of minichromosome maintenance protein 10 in medulloblastoma and its clinical implications.

BACKGROUND: Overexpression of minichromosome maintenance (MCM) proteins 2, 3, and 7 is associated with migration and invasion in medulloblastoma (MB). However, expression profiling of all prereplication complex (pre-RC) has not been addressed in MBs. PROCEDURE: We performed mRNA expression profiling of a large set of pre-RC elements in cell lines and tumor tissues of MB. RNAi technology was employed for functional studies in MB cell lines. RESULTS: Our data showed that most of the pre-RC components are significantly overexpressed in MB. Among all pre-RC mRNAs, MCM10 showed the highest level of expression (∼500- to 1,000-fold) in MB cell lines and tissues compared to the levels detected in cerebellum. In addition, RNAi silencing of MCM10 caused reduced cell proliferation and cell viability in MB cells. CONCLUSIONS: Taken together, our study reveals that the pre-RC is dysregulated in MB. In addition, MCM10, a member of this complex, is significantly overexpressed in MB and is required for tumor cell proliferation.

Potent DNA strand annealing activity associated with mouse Mcm2∼7 heterohexameric complex.

Mini-chromosome maintenance (Mcm) is a central component for DNA unwinding reaction during eukaryotic DNA replication. Mcm2∼7, each containing a conserved ATPase motif, form a six subunit-heterohexamer. Although the reconstituted Mcm2∼7-Cdc45-GINS (CMG) complex displays DNA unwinding activity, the Mcm2∼7 complex does not generally exhibit helicase activity under a normal assay condition. We detected a strong DNA strand annealing activity in the purified mouse Mcm2∼7 heterohexamer, which promotes rapid reassociation of displaced complementary single-stranded DNAs, suggesting a potential cause for its inability to exhibit DNA helicase activity. Indeed, DNA unwinding activity of Mcm2∼7 could be detected in the presence of a single-stranded DNA that is complementary to the displaced strand, which would prevent its reannealing to the template. ATPase-deficient mutations in Mcm2, 4, 5 and 6 subunits inactivated the annealing activity, while those in Mcm2 and 5 subunits alone did not. The annealing activity of Mcm2∼7 does not require Mg2+ and ATP, and is adversely inhibited by the presence of high concentration of Mg2+ and ATP while activated by similar concentrations of ADP. Our findings show that the DNA helicase activity of Mcm2∼7 may be masked by its unexpectedly strong annealing activity, and suggest potential physiological roles of strand annealing activity of Mcm during replication stress responses.

Structure of the MCM2-7 Double Hexamer and Its Implications for the Mechanistic Functions of the Mcm2-7 Complex.

The eukaryotic minichromosome maintenance 2-7 complex is the core of the inactive MCM replication licensing complex and the catalytic core of the Cdc45-MCM-GINS replicative helicase. The years of effort to determine the structure of parts or the whole of the heterohexameric complex by X-ray crystallography and conventional cryo-EM produced limited success. Modern cryo-EM technology ushered in a new era of structural biology that allowed the determination of the structure of the inactive double hexamer at an unprecedented resolution of 3.8 Å. This review will focus on the fine details observed in the Mcm2-7 double hexameric complex and their implications for the function of the Mcm2-7 hexamer in its different roles during DNA replication.

Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

Mcm10 is required for oogenesis and early embryogenesis in Drosophila.

Efficient replication of the genome and the establishment of endogenous chromatin states are processes that are essential to eukaryotic life. It is well documented that Mcm10 is intimately linked to both of these important biological processes; therefore, it is not surprising that Mcm10 is commonly misregulated in many human cancers. Most of the research regarding the biological roles of Mcm10 has been performed in single-cell or cell-free in-vitro systems. Though these systems are informative, they are unable to provide information on the cell-specific function of Mcm10 in the context of the tissue and organ systems that comprise multicellular eukaryotes. We therefore sought to identify the potential biological functions of Mcm10 in the context of a complex multicellular organism by continuing our analysis in Drosophila using three novel hypomorphic alleles. Observation of embryonic nuclear morphology and quantification of embryo hatch rates reveal that maternal loading of Mcm10 is required for embryonic nuclear stability, and suggest a role for Mcm10 post zygotic transition. Contrary to the essential nature of Mcm10 depicted in the literature, it does not appear to be required for adult viability in Drosophila if embryonic requirements are met. Although not required for adult somatic viability, analysis of fecundity and ovarian morphology in mutant females suggest that Mcm10 plays a role in maintenance of the female germline. Taken together, our results demonstrate critical roles for Mcm10 during early embryogenesis, and mark the first data linking Mcm10 to female specific reproduction in multicellular eukaryotes.

MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway.

Maintenance of genome integrity is crucial for the germline, and this is reflected by lower mutation rates in gametes than somatic cells. Germ cells at different stages employ different DNA damage response (DDR) mechanisms. In response to certain DNA repair defects, primordial germ cells (PGCs) either undergo apoptosis or delayed proliferation, although little is known about the underlying mechanisms that govern these outcomes. Here, we report genetic studies of DDR pathways that underlie germ cell depletion in mice mutant for minichromosome maintenance 9 (Mcm9), a gene that plays a role in homologous recombination repair (HRR). Germ cell depletion in these mice is a result of reduced PGC numbers both before and after they arrive in the primitive gonads. This reduction was attributable to reduced proliferation, not apoptosis, and this response was independent of ATM-CHK2-TRP53-P21 signaling. This mechanism of PGC depletion differs from that in Fancm mutants, which also display reduced PGC depletion that is partially orchestrated by the ATM-TRP53-P21 pathway. Germ cell depletion in mice doubly deficient for FANCM and MCM9 was additive, indicating that the damage caused by each mutation triggers different DDR pathways to slow the cell cycle as a means to preserve genomic integrity. genesis 53:678-684, 2015. © 2015 Wiley Periodicals, Inc.

Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.

Loading 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.

Multiple functions for Mcm2-7 ATPase motifs during replication initiation.

The Mcm2-7 replicative helicase is central to all steps of eukaryotic DNA replication. The hexameric ring of Mcm subunits forms six essential ATPases whose contributions to replication initiation remain unclear. Mcm2-7 complexes containing ATPase-motif mutations showed Mcm2-7 ATP binding and hydrolysis are required for helicase loading. Loading-defective Mcm2-7 mutant complexes were defective in initial Mcm2-7 recruitment or Cdt1 release. Comparison with Cdc6 ATPase mutants showed that Cdc6 ATP hydrolysis is not required for helicase loading but instead drives removal of Mcm2-7 complexes that cannot complete loading. A subset of Mcm2-7 ATPase-site mutants completed helicase loading but could not initiate replication. Individual mutants were defective in distinct events during helicase activation, including maintenance of DNA association, recruitment of the GINS helicase activator, and DNA unwinding. Consistent with its heterohexameric structure, our findings show that the six Mcm2-7 ATPase active sites are specialized for different functions during helicase loading and activation.

The role of the MCM2-7 helicase complex during Arabidopsis seed development.

The MINICHROMOSOME MAINTENANCE 2-7 (MCM2-7) complex, a ring-shaped heterohexamer, unwinds the DNA double helix ahead of the other replication machinery. Although there is evidence that individual components might have other roles, the essential nature of the MCM2-7 complex in DNA replication has made it difficult to uncover these. Here, we present a detailed analysis of Arabidopsis thaliana mcm2-7 mutants and reveal phenotypic differences. The MCM2-7 genes are coordinately expressed during development, although MCM7 is expressed at a higher level in the egg cell. Consistent with a role in the egg cell, heterozygous mcm7 mutants resulted in frequent ovule abortion, a phenotype that does not occur in other mcm mutants. All mutants showed a maternal effect, whereby seeds inheriting a maternal mutant allele occasionally aborted later in seed development with defects in embryo patterning, endosperm nuclear size, and cellularization, a phenotype that is variable between subunit mutants. We provide evidence that this maternal effect is due to the necessity of a maternal store of MCM protein in the central cell that is sufficient for maintaining seed viability and size in the absence of de novo MCM transcription. Reducing MCM levels using endosperm-specific RNAi constructs resulted in the up-regulation of DNA repair transcripts, consistent with the current hypothesis that excess MCM2-7 complexes are loaded during G1 phase, and are required during S phase to overcome replicative stress or DNA damage. Overall, this study demonstrates the importance of the MCM2-7 subunits during seed development and suggests that there are functional differences between the subunits.

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

Preparing to unwind.

A combination of protein crystallography and biochemistry has revealed how a ring-shaped helicase might trap a single DNA strand as the double helix melts, and before it is unwound.

MCM10: one tool for all-Integrity, maintenance and damage control.

Minichromsome maintenance protein 10 (Mcm10) is an essential replication factor that is required for the activation of the Cdc45:Mcm2-7:GINS helicase. Mcm10's ability to bind both ds and ssDNA appears vital for this function. In addition, Mcm10 interacts with multiple players at the replication fork, including DNA polymerase-α and proliferating cell nuclear antigen with which it cooperates during DNA elongation. Mcm10 lacks enzymatic function, but instead provides the replication apparatus with an oligomeric scaffold that likely acts in the coordination of DNA unwinding and DNA synthesis. Not surprisingly, loss of Mcm10 engages checkpoint, DNA repair and SUMO-dependent rescue pathways that collectively counteract replication stress and chromosome breakage. Here, we review Mcm10's structure and function and explain how it contributes to the maintenance of genome integrity.