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1.
Nat Commun ; 10(1): 4058, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492866

RESUMO

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/química , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica , Mutação , Ligação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
2.
Mol Cell ; 73(6): 1255-1266.e4, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30737186

RESUMO

Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.


Assuntos
Dano ao DNA , DNA Fúngico/genética , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Topoisomerases/genética , DNA Topoisomerases/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Cinética , Conformação de Ácido Nucleico , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
3.
J Biol Chem ; 293(27): 10524-10535, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29599286

RESUMO

Homologous recombination enables the cell to access and copy intact DNA sequence information in trans, particularly to repair DNA damage affecting both strands of the double helix. Here, we discuss the DNA transactions and enzymatic activities required for this elegantly orchestrated process in the context of the repair of DNA double-strand breaks in somatic cells. This includes homology search, DNA strand invasion, repair DNA synthesis, and restoration of intact chromosomes. Aspects of DNA topology affecting individual steps are highlighted. Overall, recombination is a dynamic pathway with multiple metastable and reversible intermediates designed to achieve DNA repair with high fidelity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Transdução de Sinais , Animais , Replicação do DNA , Humanos
4.
Cell ; 170(4): 760-773.e15, 2017 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-28781165

RESUMO

Inaccurate repair of broken chromosomes generates structural variants that can fuel evolution and inflict pathology. We describe a novel rearrangement mechanism in which translocation between intact chromosomes is induced by a lesion on a third chromosome. This multi-invasion-induced rearrangement (MIR) stems from a homologous recombination byproduct, where a broken DNA end simultaneously invades two intact donors. No homology is required between the donors, and the intervening sequence from the invading molecule is inserted at the translocation site. MIR is stimulated by increasing homology length and spatial proximity of the donors and depends on the overlapping activities of the structure-selective endonucleases Mus81-Mms4, Slx1-Slx4, and Yen1. Conversely, the 3'-flap nuclease Rad1-Rad10 and enzymes known to disrupt recombination intermediates (Sgs1-Top3-Rmi1, Srs2, and Mph1) inhibit MIR. Resolution of MIR intermediates propagates secondary chromosome breaks that frequently cause additional rearrangements. MIR features have implications for the formation of simple and complex rearrangements underlying human pathologies.


Assuntos
Cromossomos/metabolismo , Reparo do DNA , Instabilidade Genômica , Translocação Genética , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Humanos , Saccharomyces cerevisiae/genética
5.
Nucleic Acids Res ; 43(6): 3180-96, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25765654

RESUMO

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.


Assuntos
DNA Helicases/metabolismo , Reparo do DNA , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Humanos , Células MCF-7 , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutagênicos/metabolismo , Neoplasias/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo
6.
Nucleic Acids Res ; 42(10): 6511-22, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24744239

RESUMO

Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3'-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/química , Clivagem do DNA , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/química , Endonucleases/química , Endonucleases Flap/química , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química
7.
Mol Cell ; 53(3): 420-32, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24486020

RESUMO

The displacement loop (D loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, the Rad51 DNA strand exchange protein is assisted in D loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function of Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associated DNA synthesis. Here, we uncover features of D loop formation/dissociation dynamics, employing Rad51 filaments formed on ssDNAs that mimic the physiological length and structure of in vivo substrates. The Rad54 motor is activated by Rad51 bound to synapsed DNAs and guided by a ssDNA-binding domain. We present a unified model wherein Rad54 acts as an hDNA pump that drives D loop formation while simultaneously removing Rad51 from hDNA, consolidating both ATP-dependent activities of Rad54 into a single mechanistic step.


Assuntos
DNA Helicases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Recombinação Homóloga/fisiologia , Ácidos Nucleicos Heteroduplexes/metabolismo , Rad51 Recombinase/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , Modelos Genéticos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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