Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Cell Rep ; 28(10): 2673-2688.e8, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31484077

ABSTRACT

In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.


Subject(s)
Adenosine Triphosphate/metabolism , DNA Helicases/metabolism , DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Eukaryota/enzymology , Multienzyme Complexes/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Cryoelectron Microscopy , DNA/ultrastructure , DNA Helicases/chemistry , DNA Helicases/ultrastructure , Drosophila Proteins/metabolism , Drosophila melanogaster/enzymology , Hydrolysis , Models, Molecular , Protein Domains , Saccharomyces cerevisiae/metabolism
2.
Nat Commun ; 9(1): 5061, 2018 11 29.
Article in English | MEDLINE | ID: mdl-30498216

ABSTRACT

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.


Subject(s)
DNA Polymerase II/chemistry , DNA Polymerase II/metabolism , DNA Replication/physiology , DNA-Binding Proteins/metabolism , DNA/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA/chemistry , DNA/genetics , DNA Polymerase II/genetics , DNA Replication/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Tertiary
3.
Nat Commun ; 8(1): 2241, 2017 12 21.
Article in English | MEDLINE | ID: mdl-29269875

ABSTRACT

Eukaryotic origins of replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM-DNA structure with the structure of CMG-DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of replication.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Replication , DNA-Binding Proteins/ultrastructure , DNA/ultrastructure , Minichromosome Maintenance Proteins/ultrastructure , Nuclear Proteins/ultrastructure , Nucleic Acid Conformation , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Cryoelectron Microscopy , DNA/metabolism , DNA Helicases , DNA-Binding Proteins/metabolism , Holoenzymes , Image Processing, Computer-Assisted , Minichromosome Maintenance Proteins/metabolism , Nuclear Proteins/metabolism , Phosphorylation , Protein Structure, Quaternary , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism
4.
Proc Natl Acad Sci U S A ; 114(16): 4141-4146, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28373564

ABSTRACT

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.


Subject(s)
DNA Helicases/metabolism , DNA Polymerase II/metabolism , DNA Replication , Eukaryotic Cells/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , DNA Helicases/genetics , DNA Polymerase II/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics
5.
J Mol Biol ; 428(9 Pt B): 1822-32, 2016 05 08.
Article in English | MEDLINE | ID: mdl-26829220

ABSTRACT

The MCM motor of the CMG helicase powers ahead of the eukaryotic replication machinery to unwind DNA, in a process that requires ATP hydrolysis. The reconstitution of DNA replication in vitro has established the succession of events that lead to replication origin activation by the MCM and recent studies have started to elucidate the structural basis of duplex DNA unwinding. Despite the exciting progress, how the MCM translocates on DNA remains a matter of debate.


Subject(s)
DNA Helicases/chemistry , DNA Helicases/metabolism , Eukaryota/enzymology , Minichromosome Maintenance Proteins/metabolism , Adenosine Triphosphate/metabolism , DNA Replication , Eukaryota/metabolism , Hydrolysis , Models, Biological , Models, Molecular , Protein Conformation
6.
Nat Commun ; 7: 10708, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26888060

ABSTRACT

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.


Subject(s)
DNA Helicases/metabolism , DNA/metabolism , Eukaryota/enzymology , Cryoelectron Microscopy , DNA/genetics , DNA/ultrastructure , DNA Helicases/ultrastructure , DNA Replication , Eukaryota/genetics , Eukaryota/ultrastructure
7.
EMBO Rep ; 16(7): 824-35, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26071602

ABSTRACT

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1-SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Phospholipids/metabolism , Amino Acid Sequence , Binding Sites , Biological Transport , Crystallography, X-Ray , Endoplasmic Reticulum/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Mitochondrial Proteins/metabolism , Molecular Chaperones/metabolism , Molecular Sequence Data , Phospholipids/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment
SELECTION OF CITATIONS
SEARCH DETAIL
...