Initiation Of Eukaryotic DNA Replication Research Articles

Distinct RPA functions promote eukaryotic DNA replication initiation and elongation.

Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions ineukaryoticDNA replication, the DNA damage response,and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.

DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in Caenorhabditis elegans.

Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.

Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation.

CMG (Cdc45-MCM-GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae; however, in vertebrates, origin-dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell-free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N-terminal PGY and NPF motifs, and the DONSON-GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre-replicative complex, TopBP1, and kinase activities of S-CDK and DDK. Both S-CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell-free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly.

Abstract 877: Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions

Abstract Aberrant activity of cell cycle proteins is a hallmark of cancer and therefore, cell cycle regulators are considered attractive targets in cancer therapy. One such regulator, Cell division cycle 6 (Cdc6), is a key conserved cell cycle protein responsible for the initiation of eukaryotic DNA replication. Cdc6 overexpression has been observed as an early event in malignancies. Additionally, Cdc6 has also been implicated in contributing to the aggressiveness of cervical and bladder cancers. In budding yeast, Cdc6 collaborates with proteins such as Cyclin B2 (Clb2) and Cell division control protein 4 (Cdc4) to ensure the fidelity of cell division by preventing re-replication. The perfectly orchestrated sequence of events requires Cdc6 to interact with other cell cycle proteins during the correct stage of the cell cycle. During S-phase, Cyclin-dependent kinase 1 (Cdk1) regulates Cdc6 levels and Cdc6 is targeted for degradation by polyubiquination via Cdc4 (SCF-complex). In mitosis, phosphorylation by Cdk1, induces the binding of Cdc6 to Clb2-Cdk1. Clb2 binds to Cdc6’s 44PEKLQF49 motif at the Cdc6 N-terminus thereby preventing Cdc6 from licensing origins, preventing DNA re-replication, and ensuring genomic integrity. Although, it has been well studied how each Cdc6 phosphorylation site regulates protein binding, in the absence of full-length structures for many of these proteins, the interaction modes and molecular details for Cdc6’s interaction with its protein partners remain unknown. In this study, we have characterized and analyzed Cdc6’s interactions with some of its key interactors. We present a robust computational approach to understand the molecular mechanisms of Cdc6 interactions with Cdc4 and Clb2, which includes homology/ab initio modeling of the full-length structures including their intrinsically disordered regions (IDRs), and predicted interaction scenarios. Our results show that the Cdc6-Cdc4 interaction displays a dynamic shift between S-phase and mitosis. In S-phase, Cdc4 binds to the N’ phosphodegrons on Cdc6, but in mitosis, Cdc4 binding shifts to the C’ phosphodegrons. In addition, we show Clb2’s K270, within its hydrophobic patch, binds E45 in Cdc6’s 44PEKLQF49 motif. We also provide a comparison of Clb2’s interaction with Cdc6 with that of Bud3 to assess the conservation of the binding mechanism. We show that the molecular mechanism is conserved with similar residues and motifs being involved in the interaction. By exploring the molecular details underlying these protein-protein interactions, we can gain insightful information and identify interface residues that are responsible for specific protein-protein interactions. Such molecular details are critical to pave the way to the discovery of druggable targets and ultimately lead to the development of new cancer therapeutics. Citation Format: Andriele Silva Eichner, Jasmin Philip, Amy Ikui, Shaneen Singh. Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 877.

High-Throughput Screening and Molecular Dynamics Simulation of Natural Products for the Identification of Anticancer Agents against MCM7 Protein.

Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is necessary for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is linked to cellular proliferation and is accountable for critical malignancy in many cancers. Mechanistically, the suppression of MCM7 greatly lowers the cellular proliferation associated with cancer. Advances in immunotherapy have revolutionized treatments for many types of cancer. To date, no effective small molecular candidate has been found that can stop the advancement of cancer produced by the MCM7 protein. Here, we present the findings of methods that used a combination of structure-assisted drug design, high-throughput virtual screening, and simulations studies to swiftly generate lead compounds against MCM7 protein. In the current study, we designed efficient compounds that may combat all emerging cancer targeting the common MCM7 protein. For this objective, a molecular docking and molecular dynamics (MD) simulation-based virtual screening of 29,000 NPASS library was carried out. As a consequence of using specific pharmacological, physiological, and ADMET criteria, four new prevailing compounds, NPA000018, NPA000111, NPA00305, and NPA014826, were successfully selected. The MD simulations were also used for a time period of 50 ns to evaluate for stability and dynamics behavior of the compounds. Eventually, compounds NPA000111 and NPA014826 were found to be highly potent against MCM7 protein. According to our results, the selected compounds may be effective in treating certain cancer subtypes, for which additional follow-up experimental validation is recommended.

Cryo-EM structure of human hexameric MCM2-7 complex

SummaryThe central step in the initiation of eukaryotic DNA replication is the loading of the minichromosome maintenance 2–7 (MCM2-7) complex, the core of the replicative DNA helicase, onto chromatin at replication origin. Here, we reported the cryo-EM structure of endogenous human single hexameric MCM2-7 complex with a resolution at 4.4 Å, typically an open-ring hexamer with a gap between Mcm2 and Mcm5. Strikingly, further analysis revealed that human MCM2-7 can self-associate to form a loose double hexamer which potentially implies a novel mechanism underlying the MCM2-7 loading in eukaryote. The high-resolution cryo-EM structure of human MCM2-7 is critical for understanding the molecular mechanisms governing human DNA replication, especially the MCM2-7 chromatin loading and pre-replicative complex assembly.

Roles of phosphatases in eukaryotic DNA replication initiation control

In eukaryotes, the phosphorylation of replication initiation factors by protein kinases is crucial to DNA replication control. This control ensures that the genome is only copied once per cell cycle and that replication occurs in a timely manner, minimising stress. Indeed, uncontrolled DNA replication initiation causes genome instability and occurs early on in cancer development. Here we discuss the known roles of protein phosphatases in replication initiation as part of cell cycle control and the DNA damage response. We highlight how dephosphorylation ensures that DNA replication initiation events are robust, dynamic, and spatially regulated. As many kinases involved in replication control are targets for new chemotherapies, an understanding of the role of phosphatases may give critical insights into cancer treatment.

The Initiation of Eukaryotic DNA Replication.

DNA replication in eukaryotic cells initiates from large numbers of sites called replication origins. Initiation of replication from these origins must be tightly controlled to ensure the entire genome is precisely duplicated in each cell cycle. This is accomplished through the regulation of the first two steps in replication: loading and activation of the replicative DNA helicase. Here we describe what is known about the mechanism and regulation of these two reactions from a genetic, biochemical, and structural perspective, focusing on recent progress using proteins from budding yeast.

Pharmacoinformatics-based phytochemical screening for anticancer impacts of yellow sweet clover, Melilotus officinalis (Linn.) Pall

To date, much attention has been paid to phytochemicals because of their diverse pharmacological effects on a variety of diseases such as cancer. In this regard, computer-aided drug design, as a cost- and time-effective approach, is primarily applied to investigate the drug candidates before their further costly in vitro and in vivo experimental evaluations. Accordingly, different signaling pathways and proteins can be targeted using such strategies. As a key protein for the initiation of eukaryotic DNA replication, mini-chromosome maintenance complex component 7 (MCM7) overexpression is related to the initiation and progression of aggressive malignancies. The current study was conducted to identify new potential natural compounds from the yellow sweet clover, Melilotus officinalis (Linn.) Pall, by examining the potential of 40 isolated phytochemicals against MCM7 protein. A structure-based pharmacophore model to the protein active site cavity was generated and followed by virtual screening and molecular docking. Overall, four compounds were selected for further evaluation based on their binding affinities. Our analyses revealed that two novel compounds, namely rosmarinic acid (PubChem CID:5281792) and melilotigenin (PubChem CID:14059499) might be druggable and offer safe usage in human. The stability of these two protein-ligand complex structures was confirmed through molecular dynamics simulation. The findings of this study reveal the potential of these two phytochemicals to serve as anticancer agents, while further pharmacological experiments are required to confirm their effectiveness against human cancers.

Pharmacoinformatics and molecular dynamics simulation-based phytochemical screening of neem plant (Azadiractha indica) against human cancer by targeting MCM7 protein.

Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is important for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is relative to cellular proliferation and responsible for aggressive malignancy in various cancers. Mechanistically, inhibition of MCM7 significantly reduces the cellular proliferation associated with cancer. To date, no effective small molecular candidate has been identified that can block the progression of cancer induced by the MCM7 protein. Therefore, the study has been designed to identify small molecular-like natural drug candidates against aggressive malignancy associated with various cancers by targeting MCM7 protein. To identify potential compounds against the targeted protein a comprehensive in silico drug design including molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), toxicity, and molecular dynamics (MD) simulation approaches has been applied. Seventy phytochemicals isolated from the neem tree (Azadiractha indica) were retrieved and screened against MCM7 protein by using the molecular docking simulation method, where the top four compounds have been chosen for further evaluation based on their binding affinities. Analysis of ADME and toxicity properties reveals the efficacy and safety of the selected four compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed to the protein-ligand complex structure, which confirmed the stability of the selected three compounds including CAS ID:105377-74-0, CID:12308716 and CID:10505484 to the binding site of the protein. In the study, a comprehensive data screening process has performed based on the docking, ADMET properties, and MD simulation approaches, which found a good value of the selected four compounds against the targeted MCM7 protein and indicates as a promising and effective human anticancer agent.

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.

DNA Rereplication Is Susceptible to Nucleotide-Level Mutagenesis.

The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14 , a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.

Once and Only Once

The 2019 Gairdner Prize will be given to John F.X. Diffley and Bruce Stillman for their groundbreaking work on the mechanisms and control of the initiation of eukaryotic DNA replication. No two people have contributed more extensively, or over a longer period of time, to enlighten us on how our genomes replicate themselves once and only once per cell cycle.

Control of Eukaryotic DNA Replication Initiation-Mechanisms to Ensure Smooth Transitions.

DNA replication differs from most other processes in biology in that any error will irreversibly change the nature of the cellular progeny. DNA replication initiation, therefore, is exquisitely controlled. Deregulation of this control can result in over-replication characterized by repeated initiation events at the same replication origin. Over-replication induces DNA damage and causes genomic instability. The principal mechanism counteracting over-replication in eukaryotes is a division of replication initiation into two steps—licensing and firing—which are temporally separated and occur at distinct cell cycle phases. Here, we review this temporal replication control with a specific focus on mechanisms ensuring the faultless transition between licensing and firing phases.

Interaction between DUE-B and Treslin is required to load Cdc45 on chromatin in human cells

A key step in the initiation of eukaryotic DNA replication is the binding of the activator protein Cdc45 to promote MCM helicase unwinding of the origin template. We show here that the c-myc origin DNA unwinding element-binding protein, DUE-B, interacts in HeLa cells with the replication initiation protein Treslin to allow Cdc45 loading onto chromatin. The chromatin loading of DUE-B and Treslin are mutually dependent, and the DUE-B-Treslin interaction is cell cycle-regulated to peak as cells exit G1 phase prior to the initiation of replication. The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and the MCM2-7 complex, as well as for the efficient loading of Treslin, Cdc45, and TopBP1 on chromatin. These results suggest that DUE-B acts to identify origins by MCM binding and serves as a node for replication protein recruitment and Cdc45 transfer to the prereplication complex.

The mechanism of eukaryotic CMG helicase activation

The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase.

Activation of the Mcm2-7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2-7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2-7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2-7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.

DNA replication and inter-strand crosslink repair: Symmetric activation of dimeric nanomachines?

Eukaryotic DNA replication initiation and the Fanconi anemia pathway of interstrand crosslink repair both revolve around the recruitment of a set of DNA-processing factors onto a dimeric protein complex, which functions as a loading platform (MCM and FANCI-FANCD2 respectively). Here we compare and contrast the two systems, identifying a set of unresolved mechanistic questions. How is the dimeric loading platform assembled on the DNA? How can equivalent covalent modification of both factors in a dimer be achieved? Are multicomponent DNA-interacting machines built symmetrically around their dimeric loading platform? Recent biochemical reconstitution studies are starting to shed light on these issues.

Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication.

In eukaryotic DNA replication initiation, hexameric MCM (mini-chromosome maintenance) unwinds the template double-stranded DNA to form the replication fork. MCM is activated by two proteins, Cdc45 and GINS, which constitute the ‘CMG’ unwindosome complex together with the MCM core. The archaeal DNA replication system is quite similar to that of eukaryotes, but only limited knowledge about the DNA unwinding mechanism is available, from a structural point of view. Here, we describe the crystal structure of an archaeal GAN (GINS-associated nuclease) from Thermococcus kodakaraensis, the homolog of eukaryotic Cdc45, in both the free form and the complex with the C-terminal domain of the cognate Gins51 subunit (Gins51C). This first archaeal GAN structure exhibits a unique, ‘hybrid’ structure between the bacterial RecJ and the eukaryotic Cdc45. GAN possesses the conserved DHH and DHH1 domains responsible for the exonuclease activity, and an inserted CID (CMG interacting domain)-like domain structurally comparable to that in Cdc45, suggesting its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. A structural comparison of the GAN–Gins51C complex with the GINS tetramer suggests that GINS uses the mobile Gins51C as a hook to bind GAN for CMG formation.