Cdc45-MCM-GINS Research Articles

In Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast

SummaryDisassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCFDia2. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4.

The mechanism of DNA unwinding by the eukaryotic replicative helicase

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

Myc-driven chromatin accessibility regulates Cdc45 assembly into CMG helicases

Myc-driven tumorigenesis involves a non-transcriptional role for Myc in over-activating replication origins. We show here that the mechanism underlying this process involves a direct role for Myc in activation of Cdc45-MCM-GINS (CMG) helicases at Myc-targeted sites. Myc induces decondensation of higher-order chromatin at targeted sites and is required for chromatin access at a chromosomal origin. Myc-driven chromatin accessibility promotes Cdc45/GINS recruitment to resident MCMs, and activation of CMGs. Myc-Box II, which is necessary for Myc-driven transformation, is required for Myc-induced chromatin accessibility, Cdc45/GINS recruitment, and replication stimulation. Myc interactors GCN5, Tip60, and TRRAP are essential for chromatin unfolding and recruitment of Cdc45, and co-expression of GCN5 or Tip60 with MBII-deficient Myc rescues these events and promotes CMG activation. Finally, Myc and Cdc45 interact and physiologic conditions for CMG assembly require the functions of Myc, MBII, and GCN5 for Cdc45 recruitment and initiation of DNA replication.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C.elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

TRAIP is a master regulator of DNA interstrand crosslink repair.

Summary:Cells often utilize multiple pathways to repair the same DNA lesion, and pathway choice has profound implications for the fidelity of genome maintenance. DNA interstrand cross-links (ICLs) block DNA replication and transcription by covalently linking the two strands of DNA, and the cytotoxicity of ICLs is exploited for chemotherapy. In Xenopus egg extracts, replication fork collision with ICLs initiates two distinct repair pathways. The NEIL3 glycosylase can cleave the cross-link1, but if this fails, the Fanconi anemia (FA) proteins incise the phosphodiester backbone surrounding the ICL, generating a double-strand break (DSB) intermediate that is repaired by homologous recombination2. How the simpler NEIL3 pathway is prioritized over the FA pathway, which can cause genomic rearrangements, is unknown. Here, we show that the E3 ubiquitin ligase TRAIP regulates both pathways. TRAIP appears to associate with the leading edge of the replisome, ubiquitylating any protein in the replisome’s path, including the replicative DNA helicase CMG (CDC45-MCM2-7-GINS) when two replisomes converge at an ICL. In this setting, short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for CMG unloading by the p97 ATPase, enabling the FA pathway. Our results identify TRAIP as a master regulator of replisome stability and ICL repair pathway choice.

Highlights of This Issue

Three-dimensional organoid systems have emerged as potentially powerful tools to understand cancer biology, and they show promise as personalized models that can be used to guide therapeutic decisions. However, their clinical use is limited by the lack of in-depth characterization of their morphological and genetic features. In this study, Romero-Calvo and colleagues describe comprehensive histopathological, genomic and transcriptomic comparisons between patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids and their corresponding primary tumors. The organoid models recapitulate patient-specific structural and molecular features, making them potentially valuable in understanding the etiology and pathogenesis of the patient's tumor. Further, patient-specific drug sensitivities are also reproduced by the organoids, suggesting their utility as personalized models for drug testing. These findings potentiate patient-derived organoids as a promising system for precision oncology.Although the two major breast and ovarian cancer predisposing genes, BRCA1 and BRCA2, were identified over 20 years ago, new variants are still identified daily. Half of them do not show a premature stop signal, but remain classified as variants of unknown significance (VUS), which does not guide the management of the index case and her relatives. Their classification is a major challenge for the diagnosis of breast and ovarian cancer predisposition. More than 1,000 different VUS of each gene are to be classified, each constituting an independent classification effort. The functional complexity of the BRCA1 and BRCA2 macroproteins is such that the silico VUS classification tools are insufficient. This project aims to strengthen the knowledge of the impact of large sets of BRCA1 VUS on cancer risk. The current study identifies structural and functional defects in BRCA1 VUS, to improve their classification and support clinical interpretation and therapeutic selection. BRCA1/2 VUS are emblematic of the interpretation difficulties faced by geneticists with the explosion of next generation sequencing.C-X-C chemokine receptor type 7 (CXCR7), a seven-transmembrane G-protein-coupled chemokine receptor, is an androgen receptor (AR)-repressed gene in prostate cancer cells. Derepression of CXCR7 expression, after androgen deprivation therapy, is one of the survival mechanisms for castration-resistant prostate cancer (CRPC). This study suggests that CXCR7 plays more important roles than well-studied CXCR4 in CRPC development and progression. Both CXCR4 and CXCR7 are druggable targets. However, targeting CXCR7, in combination with anti-androgen, inhibits CRCP growth and potentially prevents incurable bone metastasis.Transforming growth factor ß1 (TGFß1) causes cell cycle arrest by suppressing transcriptional events and inhibiting activation of Cdc45-MCM-GINS (CMG) helicases. Notably, the retinoblastoma (Rb) tumor suppressor plays a pivotal role in both these mechanisms. However, in Rb-deficient cells, TGFß1 remains potent at eliciting growth arrest due to a previously unknown ability of TGFß1 to inhibit assembly of MCM hexamers. Overexpression of the Cdt1 oncoprotein promotes MCM assembly and abrogates this process, inducing G1-S transit in the presence of TGFß1. These results demonstrate additional redundancy in the mechanisms underlying TGFß1 growth arrest and highlight how cancer cells can overcome normal growth-regulatory signals.

Mcm10 has potent strand-annealing activity and limits translocase-mediated fork regression

The 11-subunit eukaryotic replicative helicase CMG (Cdc45, Mcm2-7, GINS) tightly binds Mcm10, an essential replication protein in all eukaryotes. Here we show that Mcm10 has a potent strand-annealing activity both alone and in complex with CMG. CMG-Mcm10 unwinds and then reanneals single strands soon after they have been unwound in vitro. Given the DNA damage and replisome instability associated with loss of Mcm10 function, we examined the effect of Mcm10 on fork regression. Fork regression requires the unwinding and pairing of newly synthesized strands, performed by a specialized class of ATP-dependent DNA translocases. We show here that Mcm10 inhibits fork regression by the well-known fork reversal enzyme SMARCAL1. We propose that Mcm10 inhibits the unwinding of nascent strands to prevent fork regression at normal unperturbed replication forks, either by binding the fork junction to form a block to SMARCAL1 or by reannealing unwound nascent strands to their parental template. Analysis of the CMG-Mcm10 complex by cross-linking mass spectrometry reveals Mcm10 interacts with six CMG subunits, with the DNA-binding region of Mcm10 on the N-face of CMG. This position on CMG places Mcm10 at the fork junction, consistent with a role in regulating fork regression.

The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC toenable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

MINA53 deficiency leads to glioblastoma cell apoptosis via inducing DNA replication stress and diminishing DNA damage response

MYC-induced nuclear antigen (MINA53) is a JmjC (jumonji C domain)-containing protein, which is highly expressed in many cancers including glioblastoma. We have revealed in our previous report that MINA53 is a poor prognostic indicator for glioblastoma patients, and knockdown of MINA53 could reduce glioblastoma malignancy. In this study, we found that MINA53 knockdown could decrease the DNA replication initiation in glioblastoma cells. Through further investigations, we revealed that MINA53 could regulate the expression of the CDC45-MCM-GINS (CMG) complex genes, which are vital for DNA replication initiation. Knockdown of MINA53 reduced the CMG genes expression and thus induced DNA replication stress and DNA damage. Furthermore, MINA53 knockdown diminished DNA damage response (DDR) by reducing the ATM/ATR-H2AX pathway activity and finally led glioblastoma cells to apoptosis and death. We further applied a genotoxic drug Doxorubicin and found that MINA53 deficiency sensitized glioblastoma cells to Doxorubicin. Our study reveals that MINA53 is involved in DNA replication initiation and DNA damage response, and provides support for MINA53 as a novel and potential therapeutic target for glioblastoma treatment.

The Mcm2-Ctf4-Polα Axis Facilitates Parental Histone H3-H4 Transfer to Lagging Strands

Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Polα axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging-strand DNA at replication forks. Mutating the conserved histone-binding domain of the Mcm2 subunit of the CMG (Cdc45-MCM-GINS) DNA helicase, which translocates along the leading-strand template, results in a marked enrichment of parental (H3-H4)2 on leading strand, due to the impairment of the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Polα primase mutants that disrupt the connection of the CMG helicase to Polα that resides on lagging-strand template. Our results support a model whereby parental (H3-H4)2 complexes displaced from nucleosomes by DNA unwinding at replication forks are transferred by the CMG-Ctf4-Polα complex to lagging-strand DNA for nucleosome assembly at the original location.

The archaeal RecJ-like proteins: nucleases and ex-nucleases with diverse roles in replication and repair.

RecJ proteins belong to the DHH superfamily of phosphoesterases that has members in all three domains of life. In bacteria, the archetypal RecJ is a 5' → 3' ssDNA exonuclease that functions in homologous recombination, base excision repair and mismatch repair, while in eukaryotes, the RecJ-like protein Cdc45 (which has lost its nuclease activity) is a key component of the CMG (Cdc45-MCM-GINS) complex, the replicative DNA helicase that unwinds double-stranded DNA at the replication fork. In archaea, database searching identifies genes encoding one or more RecJ family proteins in almost all sequenced genomes. Biochemical analysis has confirmed that some but not all of these proteins are components of archaeal CMG complexes and has revealed a surprising diversity in mode of action and substrate preference. In addition to this, some archaea encode catalytically inactive RecJ-like proteins, and others a mix of active and inactive proteins, with the inactive proteins being confined to structural roles only. Here, I summarise current knowledge of the structure and function of the archaeal RecJ-like proteins, focusing on similarities and differences between proteins from different archaeal species, between proteins within species and between the archaeal proteins and their bacterial and eukaryotic relatives. Models for RecJ-like function are described and key areas for further study highlighted.

Replication Fork Reversal during DNA Interstrand Crosslink Repair Requires CMG Unloading

SUMMARYDNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate.

Crystal Structure of Entamoeba histolytica Cdc45 Suggests a Conformational Switch that May Regulate DNA Replication

SummaryCdc45 plays a critical role at the core of the eukaryotic DNA replisome, serving as an essential scaffolding component of the replicative helicase holoenzyme Cdc45-MCM-GINS (CMG) complex. A 1.66-Å-resolution crystal structure of the full-length Cdc45 protein from Entamoeba histolytica shows a protein fold similar to that observed previously for human Cdc45 in its active conformation, featuring the overall disk-like monomer shape and intimate contacts between the N- and C-terminal DHH domains. However, the E. histolytica Cdc45 structure shows several unique features, including a distinct orientation of the C-terminal DHHA1 domain, concomitant disordering of the adjacent protruding α-helical segment implicated in DNA polymerase ɛ interactions, and a unique conformation of the GINS/Mcm5-binding loop. These structural observations collectively suggest the possibility that Cdc45 can sample multiple conformations corresponding to different functional states. We propose that such conformational switch of Cdc45 may allow regulation of protein-protein interactions important in DNA replication.

The Human Replicative Helicase, the CMG Complex, as a Target for Anti-cancer Therapy.

DNA helicases unwind or rearrange duplex DNA during replication, recombination and repair. Helicases of many pathogenic organisms such as viruses, bacteria, and protozoa have been studied as potential therapeutic targets to treat infectious diseases, and human DNA helicases as potential targets for anti-cancer therapy. DNA replication machineries perform essential tasks duplicating genome in every cell cycle, and one of the important functions of these machineries are played by DNA helicases. Replicative helicases are usually multi-subunit protein complexes, and the minimal complex active as eukaryotic replicative helicase is composed of 11 subunits, requiring a functional assembly of two subcomplexes and one protein. The hetero-hexameric MCM2-7 helicase is activated by forming a complex with Cdc45 and the hetero-tetrameric GINS complex; the Cdc45-Mcm2-7-GINS (CMG) complex. The CMG complex can be a potential target for a treatment of cancer and the feasibility of this replicative helicase as a therapeutic target has been tested recently. Several different strategies have been implemented and are under active investigations to interfere with helicase activity of the CMG complex. This review focuses on the molecular function of the CMG helicase during DNA replication and its relevance to cancers based on data published in the literature. In addition, current efforts made to identify small molecules inhibiting the CMG helicase to develop anti-cancer therapeutic strategies were summarized, with new perspectives to advance the discovery of the CMG-targeting drugs.

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism.

The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model

During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion.

The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.

CMG–Pol epsilon dynamics suggests a mechanism for the establishment of leading-strand synthesis in the eukaryotic replisome

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.