scholarly journals The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication

2002 ◽  
Vol 159 (4) ◽  
pp. 541-547 ◽  
Author(s):  
Ruth A. Van Hatten ◽  
Antonin V. Tutter ◽  
Antonia H. Holway ◽  
Alyssa M. Khederian ◽  
Johannes C. Walter ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Kinases ◽  
Origin Recognition Complex ◽  
The Other ◽  
Initiation Complex ◽  
Complex Assembly ◽  
Replicative Helicase ◽  
Parallel Pathways ◽  
Assembly Pathway ◽  
Eukaryotic Dna

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat–containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin.

scholarly journals Architecture of the yeast origin recognition complex bound to origins of DNA replication.

1997 ◽  
Vol 17 (12) ◽  
pp. 7159-7168 ◽  
Author(s):  
D G Lee ◽  
S P Bell
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Assays ◽  
Chromosomal Replication ◽  
Origins Of Replication ◽  
Eukaryotic Dna ◽  
Level Of Analysis ◽  
Origin Recognition

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.

scholarly journals The Fkh1 Forkhead Associated Domain Promotes ORC Binding to a Subset of DNA Replication Origins in Budding Yeast

2021 ◽  
Author(s):  
Timothy Hoggard ◽  
Allison J. Hollatz ◽  
Rachel Cherney ◽  
Catherine A. Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Origin Recognition Complex ◽  
Budding Yeast ◽  
Chromosomal Dna ◽  
Nucleotide Substitutions ◽  
Fha Domain ◽  
Direct Assessment ◽  
Eukaryotic Dna ◽  
Dna Replication Origins

AbstractThe pioneer event in eukaryotic DNA replication is binding of chromosomal DNA by the origin recognition complex (ORC), which directs the formation of origins, the specific chromosomal regions where DNA will be unwound for the initiation of DNA synthesis. In all eukaryotes, incompletely understood features of chromatin promote ORC-DNA binding. Here, we uncover a role for the Fkh1 (forkhead homolog) protein, and, in particular, its forkhead associated (FHA) domain in promoting ORC-origin binding and origin activity at a subset of origins in Saccharomyces cerevisiae. The majority of the FHA-dependent origins within the experimental subset examined contain a distinct Fkh1 binding site located 5’ of and proximal to their ORC sites (5’-FKH-T site). Epistasis experiments using selected FHA-dependent origins provided evidence that the FHA domain promoted origin activity through Fkh1 binding directly to this 5’ FKH-T site. Nucleotide substitutions within two of these origins that enhanced the affinity of their ORC sites for ORC bypassed these origins’ requirement for their 5’ FKH-T sites and for the FHA domain. Significantly, direct assessment of ORC-origin binding by ChIPSeq provided evidence that this mechanism affected ~25% of yeast origins. Thus, this study reveals a new mechanism to enhance ORC-origin binding in budding yeast that requires the FHA domain of the conserved cell-cycle transcription factor Fkh1.

scholarly journals Mitotic replisome disassembly in vertebrates

2018 ◽  
Author(s):  
Sara Priego Moreno ◽  
Rebecca M. Jones ◽  
Divyasree Poovathumkadavil ◽  
Agnieszka Gambus
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Replication Forks ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Egg Extract ◽  
Eukaryotic Dna ◽  
Higher Eukaryotes

ABSTRACTRecent years have brought a breakthrough in our understanding of the process of eukaryotic DNA replication termination. We have shown that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in S-phase of the cell cycle is driven through polyubiquitylation of one of the replicative helicase subunits Mcm7. Our previous work in C.elegans embryos suggested also an existence of a back-up pathway of replisome disassembly in mitosis. Here we show, that in Xenopus laevis egg extract, any replisome retained on chromatin after S-phase is indeed removed from chromatin in mitosis. This mitotic disassembly pathway depends on formation of K6 and K63 ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and activity of p97/VCP protein segregase. The mitotic replisome pathway is therefore conserved through evolution in higher eukaryotes. However, unlike in lower eukaryotes it does not require SUMO modifications. This process can also remove any helicases from chromatin, including “active” stalled ones, indicating a much wider application of this pathway than just a “back-up” for terminated helicases.

Eukaryotic DNA replication: from pre-replication complex to initiation complex

2000 ◽  
Vol 12 (6) ◽  
pp. 690-696 ◽  
Author(s):  
Haruhiko Takisawa ◽  
Satoru Mimura ◽  
Yumiko Kubota
Keyword(s):  
Dna Replication ◽  
Initiation Complex ◽  
Replication Complex ◽  
Eukaryotic Dna

scholarly journals The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

2021 ◽  
Author(s):  
Zeynep Tarcan ◽  
Divyasree Poovathumkadavil ◽  
Aggeliki Skagia ◽  
Agnieszka Gambus
Keyword(s):  
Dna Replication ◽  
Molecular Mechanism ◽  
Protein Complexes ◽  
Ubiquitin Ligases ◽  
S Phase ◽  
E3 Ubiquitin Ligases ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Cellular Processes ◽  
Eukaryotic Dna

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly in S-phase. Only Ubxn7 however facilitates efficient replisome disassembly through its interaction with both Cul2Lrr1 and p97. Our data therefore characterise Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates.

The quaternary structure of the eukaryotic DNA replication proteins Sld7 and Sld3

2015 ◽  
Vol 71 (8) ◽  
pp. 1649-1656 ◽  
Author(s):  
Hiroshi Itou ◽  
Yasuo Shirakihara ◽  
Hiroyuki Araki
Keyword(s):  
Dna Replication ◽  
Quaternary Structure ◽  
Limited Range ◽  
Replication Proteins ◽  
Replication Origins ◽  
Chromosomal Dna ◽  
Structural Domains ◽  
Replicative Helicase ◽  
Initiation Step ◽  
Eukaryotic Dna

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomes. Yeast Sld3 and its metazoan counterpart treslin are the hub proteins mediating protein associations critical for formation of the helicase. The Sld7 protein interacts with Sld3, and the complex formed is thought to regulate the function of Sld3. Although Sld7 is a non-essential DNA replication protein that is found in only a limited range of yeasts, its depletion slowed the growth of cells and caused a delay in the S phase. Recently, the Mdm2-binding protein was found to bind to treslin in humans, and its depletion causes defects in cells similar to the depletion of Sld7 in yeast, suggesting their functional relatedness and importance during the initiation step of DNA replication. Here, the crystal structure of Sld7 in complex with Sld3 is presented. Sld7 comprises two structural domains. The N-terminal domain of Sld7 binds to Sld3, and the C-terminal domains connect two Sld7 molecules in an antiparallel manner. The quaternary structure of the Sld3–Sld7 complex shown from the crystal structures appears to be suitable to activate two helicase molecules loaded onto replication origins in a head-to-head manner.

scholarly journals Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6

2020 ◽  
Vol 117 (30) ◽  
pp. 17747-17756 ◽  
Author(s):  
Zuanning Yuan ◽  
Sarah Schneider ◽  
Thomas Dodd ◽  
Alberto Riera ◽  
Lin Bai ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Origin Recognition Complex ◽  
Main Body ◽  
Structural Mechanism ◽  
Helicase Loading ◽  
M Phase ◽  
Dna Insertion ◽  
Eukaryotic Dna ◽  
Dna Replication Origins

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.

scholarly journals The mcm5-bob1 Bypass of Cdc7p/Dbf4p in DNA Replication Depends on Both Cdk1-Independent and Cdk1-Dependent Steps in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Robert A Sclafani ◽  
Marianne Tecklenburg ◽  
Angela Pierce
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Protein Kinases ◽  
Molecular Analysis ◽  
Molecular Conformation ◽  
Ectopic Expression ◽  
The Other ◽  
G1 Phase ◽  
Combined Action ◽  
Initiation Of Dna Replication

Abstract The roles in DNA replication of two distinct protein kinases, Cdc7p/Dbf4p and Cdk1p/Clb (B-type cyclin), were studied. This was accomplished through a genetic and molecular analysis of the mechanism by which the mcm5-bob1 mutation bypasses the function of the Cdc7p/Dbf4p kinase. Genetic experiments revealed that loss of either Clb5p or Clb2p cyclins suppresses the mcm5-bob1 mutation and prevents bypass. These two cyclins have distinct roles in bypass and presumably in DNA replication as overexpression of one could not complement the loss of the other. Furthermore, the ectopic expression of CLB2 in G1 phase cannot substitute for CLB5 function in bypass of Cdc7p/Dbf4p by mcm5-bob1. Molecular experiments revealed that the mcm5-bob1 mutation allows for constitutive loading of Cdc45p at early origins in arrested G1 phase cells when both kinases are inactive. A model is proposed in which the Mcm5-bob1 protein assumes a unique molecular conformation without prior action by either kinase. This conformation allows for stable binding of Cdc45p to the origin. However, DNA replication still cannot occur without the combined action of Cdk1p/Clb5p and Cdk1p/Clb2p. Thus Cdc7p and Cdk1p kinases catalyze the initiation of DNA replication at several distinct steps, of which only a subset is bypassed by the mcm5-bob1 mutation.

scholarly journals Geminin Inhibits a Late Step in the Formation of Human Pre-replicative Complexes

2014 ◽  
Vol 289 (44) ◽  
pp. 30810-30821 ◽  
Author(s):  
Min Wu ◽  
Wenyan Lu ◽  
Ruth E. Santos ◽  
Mark G. Frattini ◽  
Thomas J. Kelly
Keyword(s):  
Dna Replication ◽  
Origin Recognition Complex ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Initial Step ◽  
High Salt ◽  
Late Step ◽  
Eukaryotic Dna ◽  
Initiation Of Dna Replication

The initial step in initiation of eukaryotic DNA replication involves the assembly of pre-replicative complexes (pre-RCs) at origins of replication during the G1 phase of the cell cycle. In metazoans initiation is inhibited by the regulatory factor Geminin. We have purified the human pre-RC proteins, studied their interactions in vitro with each other and with origin DNA, and analyzed the effects of HsGeminin on formation of DNA-protein complexes. The formation of an initial complex containing the human origin recognition complex (HsORC), HsCdt1, HsCdc6, and origin DNA is cooperative, involving all possible binary interactions among the components. Maximal association of HsMCM2–7, a component of the replicative helicase, requires HsORC, HsCdc6, HsCdt1, and ATP, and is driven by interactions of HsCdt1 and HsCdc6 with multiple HsMCM2–7 subunits. Formation of stable complexes, resistant to high salt, requires ATP hydrolysis. In the absence of HsMCM proteins, HsGeminin inhibits the association of HsCdt1 with DNA or with HsORC-HsCdc6-DNA complexes. However, HsGeminin does not inhibit recruitment of HsMCM2–7 to DNA to form complexes containing all of the pre-RC proteins. In fact, HsGeminin itself is a component of such complexes, and interacts directly with the HsMcm3 and HsMcm5 subunits of HsMCM2–7, as well as with HsCdt1. Although HsGeminin does not prevent the initial formation of DNA-protein complexes containing the pre-RC proteins, it strongly inhibits the formation of stable pre-RCs that are resistant to high salt. We suggest that bound HsGeminin prevents transition of the pre-RC to a state that is competent for initiation of DNA replication.

scholarly journals How MCM loading and spreading specify eukaryotic DNA replication initiation sites

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2063 ◽  
Author(s):  
Olivier Hyrien
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Origin Recognition Complex ◽  
Cell Types ◽  
S Phase ◽  
Replication Initiation ◽  
Replication Process ◽  
Dna Replication Initiation ◽  
Eukaryotic Dna ◽  
Transcription Complexes

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

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