Disorders in the CMG helicase complex increase the proliferative capacity and delay chronological aging of budding yeast.

The replication of DNA requires specialized and intricate machinery. This machinery is known as a replisome and is highly evolutionarily conserved, from simple unicellular organisms such as yeast to human cells. The replisome comprises multiple protein complexes responsible for various steps in the replication process. One crucial component of the replisome is the Cdc45-MCM-GINS (CMG) helicase complex, which unwinds double-stranded DNA and coordinates the assembly and function of other replisome components, including DNA polymerases. The genes encoding the CMG helicase components are essential for initiating DNA replication. In this study, we aimed to investigate how the absence of one copy of the CMG complex genes in heterozygous Saccharomyces cerevisiae cells impacts the cells' physiology and aging. Our data revealed that these cells exhibited a significant reduction in transcript levels for the respective CMG helicase complex proteins, as well as disruptions in the cell cycle, extended doubling times, and alterations in their biochemical profile. Notably, this study provided the first demonstration that cells heterozygous for genes encoding subunits of the CMG helicase exhibited a significantly increased reproductive potential and delayed chronological aging. Additionally, we observed a noteworthy correlation between RNA and polysaccharide levels in yeast and their reproductive potential, as well as a correlation between fatty acid levels and cell doubling times. Our findings also shed new light on the potential utility of yeast in investigating potential therapeutic targets for cancer treatment.

Mechanism of replication origin melting nucleated by CMG helicase assembly.

The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

Structure of a dimer of the Sulfolobus solfataricus MCM N-terminal domain reveals a potential role in MCM ring opening.

Cells strongly regulate DNA replication to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea strictly control DNA-replication initiation by the regulated loading of hexameric minichromosome maintenance (MCM) rings to encircle both strands of the DNA double helix followed by regulated activation of the loaded rings such that they then encircle one DNA strand while excluding the other. Both steps involve an open/closed ring transformation, allowing DNA strands to enter or exit. Here, the crystal structure of a dimer of the N-terminal domain of Sulfolobus solfataricus MCM with an intersubunit interface that is more extensive than in closed-ring structures, while including common interactions to enable facile interconversion, is presented. It is shown that the identified interface could stabilize open MCM rings by compensating for lost interactions at an open neighbor interface and that the prior open-ring cryo-EM structure of MCM loading has a similar extended interface adjacent to its open interface.

Aberrant MCM10 SUMOylation induces genomic instability mediated by a genetic variant associated with survival of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the common gastrointestinal malignancy with an inferior prognosis outcome. DNA replication licensing aberration induced by dysregulation of minichromosome maintenance proteins (MCMs) causes genomic instability and cancer metastasis. SUMOylation modification plays a pivotal role in regulation of genomic integrity, while its dysregulation fueled by preexisting germline variants in cancers remains poorly understood. METHODS: Firstly, we conducted two-stage survival analysis consisting of an exome-wide association study in 904 ESCC samples and another independent 503 ESCC samples. Then, multipronged functional experiments were performed to illuminate the potential biological mechanisms underlying the promising variants, and MCM10 influences the ESCC progression. Finally, we tested the effects of MCM10 inhibitors on ESCC cells. RESULTS: A germline variant rs2274110 located at the exon 15 of MCM10 was identified to be significantly associated with the prognosis of ESCC patients. Individuals carrying rs2274110-AA genotypes confer a poor survival (hazard ratio = 1.61, 95% confidence interval = 1.35-1.93, p = 1.35 × 10-7 ), compared with subjects carrying rs2274110-AG/GG genotypes. Furthermore, we interestingly found that the variant can increase SUMOylation levels at K669 site (Lys[K]699Arg[R]) of MCM10 protein mediated by SUMO2/3 enzymes, which resulted in an aberrant overexpression of MCM10. Mechanistically, aberrant overexpression of MCM10 facilitated the proliferation and metastasis abilities of ESCC cells in vitro and in vivo by inducing DNA over-replication and genomic instability, providing functional evidence to support our population finding that high expression of MCM10 is extensively presented in tumor tissues of ESCC and correlated with inferior survival outcomes of multiple cancer types, including ESCC. Finally, MCM10 inhibitors Suramin and its analogues were revealed to effectively block the metastasis of ESCC cells. CONCLUSIONS: These findings not only demonstrate a potential biological mechanism between aberrant SUMOylation, genomic instability and cancer metastasis, but also provide a promising biomarker aiding in stratifying ESCC individuals with different prognosis, as well as a potential therapeutic target MCM10.

Structural mechanism for replication origin binding and remodeling by a metazoan origin recognition complex and its co-loader Cdc6.

Eukaryotic DNA replication initiation relies on the origin recognition complex (ORC), a DNA-binding ATPase that loads the Mcm2-7 replicative helicase onto replication origins. Here, we report cryo-electron microscopy (cryo-EM) structures of DNA-bound Drosophila ORC with and without the co-loader Cdc6. These structures reveal that Orc1 and Orc4 constitute the primary DNA binding site in the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. A loop region near the catalytic Walker B motif of Orc1 directly contacts DNA, allosterically coupling DNA binding to ORC's ATPase site. Correlating structural and biochemical data show that DNA sequence modulates DNA binding and remodeling by ORC, and that DNA bending promotes Mcm2-7 loading in vitro. Together, these findings explain the distinct DNA sequence-dependencies of metazoan and S. cerevisiae initiators in origin recognition and support a model in which DNA geometry and bendability contribute to Mcm2-7 loading site selection in metazoans.

CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes.

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.

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.

The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance.

There are several DNA helicases involved in seemingly overlapping aspects of homologous and homoeologous recombination. Mutations of many of these helicases are directly implicated in genetic diseases including cancer, rapid aging, and infertility. MCM8/9 are recent additions to the catalog of helicases involved in recombination, and so far, the evidence is sparse, making assignment of function difficult. Mutations in MCM8/9 correlate principally with primary ovarian failure/insufficiency (POF/POI) and infertility indicating a meiotic defect. However, they also act when replication forks collapse/break shuttling products into mitotic recombination and several mutations are found in various somatic cancers. This review puts MCM8/9 in context with other replication and recombination helicases to narrow down its genomic maintenance role. We discuss the known structure/function relationship, the mutational spectrum, and dissect the available cellular and organismal data to better define its role in recombination.

The High-Affinity Interaction between ORC and DNA that Is Required for Replication Licensing Is Inhibited by 2-Arylquinolin-4-Amines.

In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.

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.

Open-ringed structure of the Cdt1-Mcm2-7 complex as a precursor of the MCM double hexamer.

The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.

Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA.

DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.

Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23(Nterm).

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at λ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23(Nterm), a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23(Nterm) has an expected Bijvoet ratio〈|Fanom|〉/〈F〉of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parameters a = b = 61.2, c = 151.5 Å, and belonged to space group P43. The refined structure to 3.1 Å resolution has an R factor of 18.7% and an Rfree of 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.

Insights into the Initiation of Eukaryotic DNA Replication.

The initiation of DNA replication is a highly regulated event in eukaryotic cells to ensure that the entire genome is copied once and only once during S phase. The primary target of cellular regulation of eukaryotic DNA replication initiation is the assembly and activation of the replication fork helicase, the 11-subunit assembly that unwinds DNA at a replication fork. The replication fork helicase, called CMG for Cdc45-Mcm2-7, and GINS, assembles in S phase from the constituent Cdc45, Mcm2-7, and GINS proteins. The assembly and activation of the CMG replication fork helicase during S phase is governed by 2 S-phase specific kinases, CDK and DDK. CDK stimulates the interaction between Sld2, Sld3, and Dpb11, 3 initiation factors that are each required for the initiation of DNA replication. DDK, on the other hand, phosphorylates the Mcm2, Mcm4, and Mcm6 subunits of the Mcm2-7 complex. Sld3 recruits Cdc45 to Mcm2-7 in a manner that depends on DDK, and recent work suggests that Sld3 binds directly to Mcm2-7 and also to single-stranded DNA. Furthermore, recent work demonstrates that Sld3 and its human homolog Treslin substantially stimulate DDK phosphorylation of Mcm2. These data suggest that the initiation factor Sld3/Treslin coordinates the assembly and activation of the eukaryotic replication fork helicase by recruiting Cdc45 to Mcm2-7, stimulating DDK phosphorylation of Mcm2, and binding directly to single-stranded DNA as the origin is melted.

A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells.

Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is sensitive to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among well-characterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small- and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.

MCM ring hexamerization is a prerequisite for DNA-binding.

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus.

The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. While PtMCM hydrolyzes ATP in DNA-independent manner, it displays very poor ability to unwind DNA independently, and then too only under acidic conditions. The protein exists stably in complex with PtGINS in whole cell lysates, interacting directly with PtGINS under neutral and acidic conditions. GINS strongly activates MCM helicase activity, but only at low pH. In consonance with this, PtGINS activates PtMCM-mediated ATP hydrolysis only at low pH, with the amount of ATP hydrolyzed during the helicase reaction increasing more than fifty-fold in the presence of GINS. While the stimulation of MCM-mediated helicase activity by GINS has been reported in MCMs from P.furiosus, T.kodakarensis, and very recently, T.acidophilum, to the best of our knowledge, this is the first report of an MCM helicase demonstrating DNA unwinding activity only at such acidic pH, across all archaea and eukaryotes. PtGINS may induce/stabilize a conducive conformation of PtMCM under acidic conditions, favouring PtMCM-mediated DNA unwinding coupled to ATP hydrolysis. Our findings underscore the existence of divergent modes of replication regulation among archaea and the importance of investigating replication events in more archaeal organisms.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex.

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain.