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1.
Science ; 384(6691): 119-124, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38484038

RESUMO

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.


Assuntos
Cromátides , Coesinas , Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Troca de Cromátide Irmã , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Coesinas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
2.
Nat Commun ; 14(1): 8339, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097584

RESUMO

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/metabolismo , Origem de Replicação , Bactérias/genética , DNA , DNA de Cadeia Simples/genética , DNA Bacteriano/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo
3.
Mol Cell ; 83(16): 2925-2940.e8, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37499663

RESUMO

Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.


Assuntos
Proteína BRCA2 , Rad51 Recombinase , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA de Cadeia Simples/genética , DNA/metabolismo , Reparo do DNA , Ligação Proteica
4.
Nat Commun ; 13(1): 2416, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35504909

RESUMO

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.


Assuntos
DNA Viral , Integrases , Animais , Humanos , Domínio Catalítico , DNA Viral/metabolismo , Integrases/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Modelos Moleculares , Retroviridae/genética , Ovinos/genética , Integração Viral
5.
Essays Biochem ; 65(1): 17-26, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33438722

RESUMO

Cell-free extracts from Xenopus laevis eggs are a model system for studying chromosome biology. Xenopus egg extracts can be synchronised in different cell cycle stages, making them useful for studying DNA replication, DNA repair and chromosome organisation. Combining single-molecule approaches with egg extracts is an exciting development being used to reveal molecular mechanisms that are difficult to study using conventional approaches. Fluorescence-based single-molecule imaging of surface-tethered DNAs has been used to visualise labelled protein movements on stretched DNA, the dynamics of DNA-protein complexes and extract-dependent structural rearrangement of stained DNA. Force-based single-molecule techniques are an alternative approach to measure mechanics of DNA and proteins. In this essay, the details of these single-molecule techniques, and the insights into chromosome biology they provide, will be discussed.


Assuntos
Replicação do DNA , Oócitos , Animais , Biologia , Cromossomos , Xenopus laevis/genética , Xenopus laevis/metabolismo
6.
Sci Rep ; 10(1): 12504, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719468

RESUMO

Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.


Assuntos
DNA/química , Imageamento Tridimensional , Microscopia de Fluorescência , Imagem Óptica , Cromossomos/metabolismo , Humanos , Células Jurkat , Cinética , Metáfase , Ligação Proteica
7.
Nat Commun ; 11(1): 3713, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709841

RESUMO

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA/química , DNA/metabolismo , Animais , Bacteriófago T4 , Microscopia Crioeletrônica , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Escherichia coli/genética
8.
Cell Rep ; 28(10): 2673-2688.e8, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484077

RESUMO

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.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Eucariotos/enzimologia , Complexos Multienzimáticos/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Hidrólise , Modelos Moleculares , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo
9.
Nat Commun ; 10(1): 2159, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089141

RESUMO

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.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Modelos Moleculares , DNA Helicases/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Fenômenos Magnéticos , Pinças Ópticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
10.
Cell Rep ; 26(8): 2113-2125.e6, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30784593

RESUMO

Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.


Assuntos
DNA Helicases/química , Replicação do DNA , Animais , Linhagem Celular , DNA/química , DNA/metabolismo , DNA Helicases/metabolismo , Cinética , Ligação Proteica , Domínios Proteicos , Spodoptera , Xenopus laevis
11.
Cell ; 159(2): 346-57, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25303529

RESUMO

DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.


Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Replicação do DNA , Animais , Extratos Celulares/química , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Óvulo/química , Xenopus
12.
Nat Struct Mol Biol ; 21(1): 20-5, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24389553

RESUMO

In G1, two copies of the MCM2-7 helicase are recruited to each origin of replication. Whereas recruitment of the first MCM2-7 is likely to be analogous to the loading of sliding clamps around DNA, how the second MCM2-7 complex is recruited is highly contentious. Here, we argue that MCM2-7 loading involves specific modifications to the clamp-loading reaction and propose that the first and second MCM2-7 molecules are loaded via similar mechanisms.


Assuntos
DNA/química , Complexo de Reconhecimento de Origem , Proteínas de Manutenção de Minicromossomo/metabolismo
13.
Nature ; 492(7428): 205-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23201686

RESUMO

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (known as steric exclusion). By contrast, large T antigen, the replicative DNA helicase of the simian virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here we use single-molecule and ensemble assays to show that large T antigen assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3'-to-5' direction. Unexpectedly, large T antigen unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the large T antigen ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms, and reveal a new level of plasticity in the interactions of replicative helicases with DNA damage.


Assuntos
DNA Helicases/metabolismo , Vírus 40 dos Símios/enzimologia , Antígenos Virais de Tumores/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Origem de Replicação/fisiologia , Proteínas Virais/metabolismo
14.
Methods ; 57(2): 179-86, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22503776

RESUMO

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.


Assuntos
Extratos Celulares/genética , Replicação do DNA , DNA Viral/biossíntese , Ácidos Nucleicos Imobilizados/biossíntese , Oócitos/citologia , Animais , Sequência de Bases , Separação Celular , DNA Viral/química , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Ácidos Nucleicos Imobilizados/química , Técnicas Analíticas Microfluídicas/instrumentação , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Coloração e Rotulagem , Xenopus laevis
15.
Cell ; 146(6): 931-41, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21925316

RESUMO

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA/metabolismo , Xenopus/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , Modelos Biológicos , Fase S
16.
Mol Cell ; 40(5): 834-40, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21145490

RESUMO

The duplication of eukaryotic genomes involves the replication of DNA from multiple origins of replication. In S phase, two sister replisomes assemble at each active origin, and they replicate DNA in opposite directions. Little is known about the functional relationship between sister replisomes. Some data imply that they travel away from one another and thus function independently. Alternatively, sister replisomes may form a stationary, functional unit that draws parental DNA toward itself. If this "double replisome" model is correct, a constrained DNA molecule should not undergo replication. To test this prediction, lambda DNA was stretched and immobilized at both ends within a microfluidic flow cell. Upon exposure to Xenopus egg extracts, this DNA underwent extensive replication by a single pair of diverging replisomes. The data show that there is no obligatory coupling between sister replisomes and, together with other studies, imply that genome duplication involves autonomously functioning replisomes.


Assuntos
Replicação do DNA , Animais , Genoma , Origem de Replicação , Fase S , Xenopus
17.
Proc Natl Acad Sci U S A ; 105(16): 6016-21, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18427114

RESUMO

In vivo studies suggest that centromeric protein E (CENP-E), a kinesin-7 family member, plays a key role in the movement of chromosomes toward the metaphase plate during mitosis. How CENP-E accomplishes this crucial task, however, is not clear. Here we present single-molecule measurements of CENP-E that demonstrate that this motor moves processively toward the plus end of microtubules, with an average run length of 2.6 +/- 0.2 mum, in a hand-over-hand fashion, taking 8-nm steps with a stall force of 6 +/- 0.1 pN. The ATP dependence of motor velocity obeys Michaelis-Menten kinetics with K(M,ATP) = 35 +/- 5 muM. All of these features are remarkably similar to those for kinesin-1-a highly processive transport motor. We, therefore, propose that CENP-E transports chromosomes in a manner analogous to how kinesin-1 transports cytoplasmic vesicles.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Mitose , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Cromossômicas não Histona/química , Cinesinas/química , Cinética , Microtúbulos/química , Xenopus
18.
J Chem Phys ; 124(21): 214503, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16774419

RESUMO

Employing frequency-dependent dielectric susceptibility we characterize the aging in two supercooled liquids, sorbitol and xylitol, below their calorimetric glass transition temperatures. In addition to the alpha relaxation that tracks the structural dynamics, the susceptibility of both liquids possesses a secondary Johari-Goldstein relaxation at higher frequencies. Following a quench through the glass transition, the susceptibility slowly approaches the equilibrium behavior. For both liquids, the magnitude of the Johari-Goldstein relaxation displays a dependence on the time since the quench, or aging time, that is quantitatively very similar to the age dependence of the alpha peak frequency. The Johari-Goldstein relaxation time remains constant during aging for sorbitol while it decreases slightly with age for xylitol. Hence, one cannot sensibly assign a fictive temperature to the Johari-Goldstein relaxation. This behavior contrasts with that of liquids lacking distinct Johari-Goldstein peaks for which the excess wing of the alpha peak tracks the main part of the peak during aging, enabling the assignment of a single fictive temperature to the entire spectrum. The aging behavior of the Johari-Goldstein relaxation time further calls into question the possibility that the relaxation time possesses stronger temperature dependence in equilibrium than is observed in the out-of-equilibrium state below the glass transition.

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