Publications
57 results found
Saleh A, Noguchi Y, Aramayo R, et al., 2022, The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer, Nature Communications, Vol: 13, ISSN: 2041-1723
The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
Feng X, Noguchi Y, Barbon M, et al., 2021, The structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6, Nature Communications, Vol: 12, ISSN: 2041-1723
The Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.
Yuan Z, Schneider S, Dodd T, et al., 2021, Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6 (vol 117, pg 17747, 2020), Proceedings of the National Academy of Sciences of the United States of America, Vol: 118, Pages: 1-1, ISSN: 0027-8424
Hu Y, Tareen A, Sheu Y-J, et al., 2020, Evolution of DNA replication origin specification and gene silencing mechanisms, Nature Communications, Vol: 11, ISSN: 2041-1723
DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
Yuan Z, Schneider S, Dodd T, et al., 2020, Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 17747-17756, ISSN: 0027-8424
DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC–Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC–Cdc6–Cdt1–Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC–Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In “semi-attached OCCM,” the Mcm3 and Mcm7 WHDs latch onto ORC–Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In “pre-insertion OCCM,” the main body of Mcm2-7 docks onto ORC–Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2–Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.
Hu Y, Tareen A, Sheu Y-J, et al., 2020, Evolution of DNA replication origin specification and gene silencing mechanisms, Publisher: Cold Spring Harbor Laboratory
DNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
Roman-Trufero M, Ito CM, Pedebos C, et al., 2020, Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3, Molecular Biology and Evolution, Vol: 37, Pages: 1986-2001, ISSN: 0737-4038
Genetic variation in the enzymes that catalyse post-translational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signalling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme, which acts as a ubiquitin donor for E3 ligases that catalyse ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologues to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse ES cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal mutation in mice. When mutant S138A ESCs were differentiated into extra-embryonic primitive endoderm (PrE), levels of the PDGFRα and FGFR1 receptor tyrosine kinases (RTKs) were reduced and PreE differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the RTKs. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.
Roman-Trufero M, Ito CM, Pedebos C, et al., 2020, Evolution of an amniote-specific mechanism for modulating ubiquitin signalling via phosphoregulation of the E2 enzyme UBE2D3, Molecular Biology and Evolution, ISSN: 0737-4038
<jats:title>Abstract</jats:title><jats:p>Genetic variation in the enzymes that catalyse post-translational modification of proteins is a potentially important source of phenotypic variation during evolution. Ubiquitination is one such modification that affects turnover of virtually all of the proteins in the cell in addition to roles in signalling and epigenetic regulation. UBE2D3 is a promiscuous E2 enzyme that acts as a ubiquitin donor for E3 ligases that catalyse ubiquitination of developmentally important proteins. We have used protein sequence comparison of UBE2D3 orthologues to identify a position in the C-terminal α-helical region of UBE2D3 that is occupied by a conserved serine in amniotes and by alanine in anamniote vertebrate and invertebrate lineages. Acquisition of the serine (S138) in the common ancestor to modern amniotes created a phosphorylation site for Aurora B. Phosphorylation of S138 disrupts the structure of UBE2D3 and reduces the level of the protein in mouse ES cells (ESCs). Substitution of S138 with the anamniote alanine (S138A) increases the level of UBE2D3 in ESCs as well as being a gain of function early embryonic lethal in mice. When mutant S138A ESCs were differentiated into extra-embryonic primitive endoderm (PrE), levels of the PDGFRα and FGFR1 receptor tyrosine kinases (RTKs) were reduced and PreE differentiation was compromised. Proximity ligation analysis showed increased interaction between UBE2D3 and the E3 ligase CBL and between CBL and the RTKs. Our results identify a sequence change that altered the ubiquitination landscape at the base of the amniote lineage with potential effects on amniote biology and evolution.</jats:p>
Mendes ML, Fischer L, Chen ZA, et al., 2019, An integrated workflow for crosslinking mass spectrometry., Mol Syst Biol, Vol: 15
We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
Mendes ML, Fischer L, Chen ZA, et al., 2019, An integrated workflow for crosslinking mass spectrometry, Molecular Systems Biology, ISSN: 1744-4292
<jats:p>We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software Xi. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.</jats:p>
Yu Y, Schleich K, Yue B, et al., 2018, Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma (vol 33, pg 322, 2018), CANCER CELL, Vol: 33, Pages: 785-785, ISSN: 1535-6108
Yu Y, Schleich K, Yue B, et al., 2018, Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma., Cancer Cell, Vol: 33, Pages: 322-336.e8, ISSN: 1535-6108
Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.
Noguchi Y, yuan Z, Bai L, et al., 2017, Cryo-EM structure of Mcm2-7 double-hexamer on DNA suggests a lagging strand DNA extrusion model, Proceedings of the National Academy of Sciences, Vol: 114, Pages: E9529-E9538, ISSN: 0027-8424
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.
Riera A, Barbon M, Noguchi Y, et al., 2017, From structure to mechanism – understanding initiation of DNA replication, Genes & Development, Vol: 31, Pages: 1073-1088, ISSN: 1549-5477
DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2–7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.
Yuan Z, Riera A, Bai L, et al., 2017, Structural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1, Nature Structural & Molecular Biology, Vol: 24, Pages: 316-324, ISSN: 1545-9993
To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an a-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.
Speck C, Riera A, Yuan Z, et al., 2016, Key mechanism in the loading and activation of the replicative helicase MCM2-7, 41st FEBS Congress on Molecular and Systems Biology for a Better Life, Publisher: WILEY-BLACKWELL, Pages: 12-12, ISSN: 1742-464X
Tognetti S, Speck C, 2016, Replicating repetitive DNA, Nature Cell Biology, Vol: 18, Pages: 593-594, ISSN: 1476-4679
The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress.
Speck C, 2016, Exceeding the limits - Cdc45 overexpression turns bad, Cell Cycle, Vol: 15, Pages: 1809-1810, ISSN: 1551-4005
Sun J, Yuan Z, Stillman B, et al., 2016, Structure and function studies of replication initiation factors, The Initiation of DNA Replication in Eukaryotes, Pages: 427-441, ISBN: 9783319246949
We have used negative stain EM and cryo-EM to visualize step by step the replication initiation events in S. cerevisiae, as the process is driven forward by the interplay of a dozen or so macromolecular initiation factors, leading to the establishment of pre-replication complexes (pre-RC) at each origin of DNA replication. This work took advantage of our ability to reconstitute the Mcm2-7 loading reaction with purified proteins. We determined the architecture of several previously known replication initiation complexes such as ORC, ORC-Cdc6 on DNA, and the Mcm2-7 double hexamer. We also captured by EM reaction intermediates such as the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) and the ORC-Cdc6-Mcm2-7-Mcm2-7 (OCMM) that had evaded previous biochemical identification. In this chapter, we describe what we have learnt about the structure and interaction with origin DNA of the replication initiators. We further discuss what may be expected in the coming years as cryo-EM is becoming a near-atomic resolution structural tool, thanks to the recent advent of the direct electron detector.
Riera A, Speck C, 2016, Licensing of replication origins, The Initiation of DNA Replication in Eukaryotes, Pages: 189-211, ISBN: 9783319246949
All living organisms need to duplicate their genetic material prior to cell division in order to maintain genomic-stability. Cells have evolved sophisticated DNA replication mechanisms to ensure that this process is as faithful as possible. Eukaryotic initiation of DNA replication is a two-step process, where the replicative DNA helicase becomes loaded onto DNA to license DNA replication during late M-phase of the cell cycle prior to helicase-activation in S-phase. Importantly, helicase loading is entirely blocked in S-phase, which is a crucial regulatory mechanism that hinders re-replication of DNA and is crucial for genomic stability. Moreover, multiple copies of the replicative helicase become loaded at each origin to serve as backup-helicases in case a fork becomes terminally arrested. For these reasons it is imperative that helicase loading is as efficient as possible. MCM2-7 represent the core of the replicative helicase, which becomes loaded in an ATP-hydrolysis-dependent process as a double-hexamer onto double-stranded DNA. Current data suggest a model where ORC, Cdc6, and Cdt1 load in a stepwise process the MCM2-7 double-hexamer onto DNA. In this review we discuss the emerging mechanism of ATP-hydrolysis-driven helicase loading, the regulation of this process, and the structure and function of the MCM2-7 double-hexamer.
Herrera MC, Tognetti S, Riera A, et al., 2015, A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase, Nucleic Acids Research, Vol: 43, Pages: 10238-10250, ISSN: 1362-4962
During G1-phase of the cell-cycle the replicative MCM2-7 helicase becomes loaded onto DNA into prereplicativecomplexes (pre-RCs), resulting in MCM2-7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number ofhelicase-activation factors the assembly of a Cdc45-MCM2-7-GINS (CMG) complex. However, in theabsence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2-7double-hexamer represents a very large interaction surface. Currently it is unclear what mechanismsrestricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. Wedeveloped an advanced reconstituted-system to study helicase activation in-solution and discoveredthat individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. WhenSld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactionsrestrict complex assembly. DDK stabilizes the Sld3/Sld2-pre-RC complex, but the complex is only shortlived,indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45-pre-RC can form in the presenceof DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicaseactivation is regulated by competitive and cooperative interactions, which restrict illegitimate complexformation and direct limiting helicase-activation factors into pre-initiation complexes.
Speck C, 2015, Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication, eLife, Vol: 4, ISSN: 2050-084X
To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.
Riera A, Speck C, 2014, Opening the gate to DNA replication, Cell Cycle, Vol: 14, Pages: 6-8, ISSN: 1551-4005
Silva N, Ferrandiz N, Barroso C, et al., 2014, The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression, Developmental Cell, Vol: 31, Pages: 503-511, ISSN: 1534-5807
Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, andpoorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint.
Sun J, Fernandez-Cid A, Riera A, et al., 2014, Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function, Genes and Development, Vol: 28, Pages: 2291-2303, ISSN: 0890-9369
Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2–7 (minichromosome maintenance proteins 2–7) double hexamer. During S phase, each Mcm2–7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC–Cdc6 function to recruit a single Cdt1–Mcm2–7 heptamer to replication origins prior to Cdt1 release and ORC–Cdc6–Mcm2–7 complex formation, but how the second Mcm2–7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC–Cdc6–Mcm2–7 complex and an ORC–Cdc6–Mcm2–7–Mcm2–7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2–7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2–7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
Tognetti S, Riera A, Speck C, 2014, Switch on the engine: how the eukaryotic replicative helicase MCM2–7 becomes activated, Chromosoma, Vol: 124, Pages: 13-26, ISSN: 1432-0886
A crucial step during eukaryotic initiation of DNA replication is the correct loading and activation of the replicative DNA helicase, which ensures that each replication origin fires only once. Unregulated DNA helicase loading and activation, as it occurs in cancer, can cause severe DNA damage and genomic instability. The essential mini-chromosome maintenance proteins 2–7 (MCM2–7) represent the core of the eukaryotic replicative helicase that is loaded at DNA replication origins during G1-phase of the cell cycle. The MCM2–7 helicase activity, however, is only triggered during S-phase once the holo-helicase Cdc45-MCM2–7-GINS (CMG) has been formed. A large number of factors and several kinases interact and contribute to CMG formation and helicase activation, though the exact mechanisms remain unclear. Crucially, upon DNA damage, this reaction is temporarily halted to ensure genome integrity. Here, we review the current understanding of helicase activation; we focus on protein interactions during CMG formation, discuss structural changes during helicase activation, and outline similarities and differences of the prokaryotic and eukaryotic helicase activation process.
Samel SA, Fernandez-Cid A, Sun J, et al., 2014, A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA, Genes and Development, Vol: 28, Pages: 1653-1666, ISSN: 0890-9369
The regulated loading of the replicative helicase minichromosome maintenance proteins 2–7 (MCM2–7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2–7 hexamers into one double hexamer around dsDNA. Although the MCM2–7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2–7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC–Cdc6–Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2–7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2–7 complex formation prior to MCM2–7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.
Stillman B, Speck C, Li H, 2014, Biochemical studies on replication of the genome in eukaryotes, Experimental Biology Meeting, Publisher: FEDERATION AMER SOC EXP BIOL, ISSN: 0892-6638
Evrin C, Fernandez-Cid A, Riera A, et al., 2014, The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 2257-2269, ISSN: 0305-1048
- Author Web Link
- Cite
- Citations: 24
Riera A, Tognetti S, Speck C, 2014, Helicase loading: How to build a MCM2-7 double-hexamer, Seminars in Cell and Developmental Biology, Vol: 30, Pages: 104-109, ISSN: 1084-9521
A central step in eukaryotic initiation of DNA replication is the loading of the helicase at replication origins, misregulation of this reaction leads to DNA damage and genome instability. Here we discuss how the helicase becomes recruited to origins and loaded into a double-hexamer around double-stranded DNA. We specifically describe the individual steps in complex assembly and explain how this process is regulated to maintain genome stability. Structural analysis of the helicase loader and the helicase has provided key insights into the process of double-hexamer formation. A structural comparison of the bacterial and eukaryotic system suggests a mechanism of helicase loading. © 2014 Elsevier Ltd.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.