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1.
Nat Commun ; 13(1): 2248, 2022 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-35473934

RESUMO

Bloom syndrome (BS) is associated with a profoundly increased cancer risk and is caused by mutations in the Bloom helicase (BLM). BLM is involved in the nucleolytic processing of the ends of DNA double-strand breaks (DSBs), to yield long 3' ssDNA tails that serve as the substrate for break repair by homologous recombination (HR). Here, we use single-molecule imaging to demonstrate that BLM mediates formation of large ssDNA loops during DNA end processing. A BLM mutant lacking the N-terminal domain (NTD) retains vigorous in vitro end processing activity but fails to generate ssDNA loops. This same mutant supports DSB end processing in cells, however, these cells do not form RAD51 DNA repair foci and the processed DSBs are channeled into synthesis-dependent strand annealing (SSA) instead of HR-mediated repair, consistent with a defect in RAD51 filament formation. Together, our results provide insights into BLM functions during homologous recombination.


Assuntos
DNA de Cadeia Simples , RecQ Helicases , DNA/genética , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35042797

RESUMO

Srs2 is a superfamily 1 (SF1) helicase that participates in several pathways necessary for the repair of damaged DNA. Srs2 regulates formation of early homologous recombination (HR) intermediates by actively removing the recombinase Rad51 from single-stranded DNA (ssDNA). It is not known whether and how Srs2 itself is down-regulated to allow for timely HR progression. Rad54 and Rdh54 are two closely related superfamily 2 (SF2) motor proteins that promote the formation of Rad51-dependent recombination intermediates. Rad54 and Rdh54 bind tightly to Rad51-ssDNA and act downstream of Srs2, suggesting that they may affect the ability of Srs2 to dismantle Rad51 filaments. Here, we used DNA curtains to determine whether Rad54 and Rdh54 alter the ability of Srs2 to disrupt Rad51 filaments. We show that Rad54 and Rdh54 act synergistically to greatly restrict the antirecombinase activity of Srs2. Our findings suggest that Srs2 may be accorded only a limited time window to act and that Rad54 and Rdh54 fulfill a role of prorecombinogenic licensing factors.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/fisiologia , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/fisiologia , DNA Topoisomerases/fisiologia , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Ligação Proteica/genética , Rad51 Recombinase/metabolismo , Rad51 Recombinase/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia
3.
Nucleic Acids Res ; 50(2): 952-961, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34967418

RESUMO

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3'-to-5' translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Mycobacterium/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Endonucleases , Ligação Proteica , Relação Estrutura-Atividade
4.
Front Cell Dev Biol ; 9: 745311, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34869333

RESUMO

DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.

6.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
7.
Methods Enzymol ; 661: 343-362, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34776219

RESUMO

Homologous recombination (HR) is a conserved mechanism essential for the accurate repair of DNA double stranded breaks and the exchange of genetic information during meiosis. The key steps in HR are carried out by the RecA/Rad51 class of recombinases, which form a helical filament on single-stranded DNA (ssDNA) and catalyze homology search and strand exchange with a complementary duplex DNA target. In eukaryotes, assembly of the Rad51-ssDNA filament requires regulatory factors called mediators, including Rad51 paralogs. A mechanistic understanding of the role of Rad51 paralogs in HR has been hampered by the transient and diverse nature of intermediates formed with the Rad51-ssDNA filament, which cannot be resolved by traditional ensemble methods. The biochemical characterization of Rad51 paralogs, including the S. cerevisiae complex Rad55-Rad57 has also been limited by their propensity to aggregate. Here we describe the preparation of monodisperse GFP-tagged Rad55-Rad57 complex and the methodology for its analysis in our single-molecule DNA curtain assay.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Genes (Basel) ; 12(9)2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34573298

RESUMO

Helicases are enzymes that convert the chemical energy stored in ATP into mechanical work, allowing them to move along and manipulate nucleic acids. The helicase superfamily 1 (Sf1) is one of the largest subgroups of helicases and they are required for a range of cellular activities across all domains of life. Sf1 helicases can be further subdivided into two classes called the Sf1a and Sf1b helicases, which move in opposite directions on nucleic acids. The results of this movement can range from the separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. Here, we describe the characteristics of the Sf1a helicase Srs2 and the Sf1b helicase Pif1, both from the model organism Saccharomyces cerevisiae, focusing on the roles that they play in homologous recombination, a DNA repair pathway that is necessary for maintaining genome integrity.


Assuntos
Proteínas de Saccharomyces cerevisiae
9.
Genes (Basel) ; 12(9)2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34573372

RESUMO

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55-Rad57 in HR.


Assuntos
Adenosina Trifosfatases/fisiologia , Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Recombinação Homóloga , Proteínas de Saccharomyces cerevisiae/fisiologia , Regulação Fúngica da Expressão Gênica , Complexos Multiproteicos , Mutação , Saccharomyces cerevisiae/genética , Imagem Individual de Molécula
10.
Methods Mol Biol ; 2281: 193-207, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847959

RESUMO

Homologous recombination (HR) is a highly conserved DNA repair pathway required for the accurate repair of DNA double-stranded breaks. DNA recombination is catalyzed by the RecA/Rad51 family of proteins, which are conserved from bacteria to humans. The key intermediate catalyzing DNA recombination is the presynaptic complex (PSC), which is a helical filament comprised of Rad51-bound single-stranded DNA (ssDNA). Multiple cellular factors either promote or downregulate PSC activity, and a fine balance between such regulators is required for the proper regulation of HR and maintenance of genomic integrity. However, dissecting the complex mechanisms regulating PSC activity has been a challenge using traditional ensemble methods due to the transient and dynamic nature of recombination intermediates. We have developed a single-molecule assay called ssDNA curtains that allows us to visualize individual DNA intermediates in real-time, using total internal reflection microscopy (TIRFM). This assay has allowed us to study many aspects of HR regulation that involve complex and heterogenous reaction intermediates. Here we describe the procedure for a basic ssDNA curtain assay to study PSC filament dynamics, and explain how to process and analyze the resulting data.


Assuntos
DNA de Cadeia Simples/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , DNA Fúngico/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Microscopia de Interferência , Proteínas de Saccharomyces cerevisiae/genética
11.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836607

RESUMO

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.


Assuntos
DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Quebras de DNA de Cadeia Dupla , DNA Helicases/química , DNA Helicases/genética , Reparo do DNA , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Hidrólise , Mutação , Mycobacterium/enzimologia , Mycobacterium/genética , Ligação Proteica , Domínios Proteicos
12.
Trends Genet ; 37(7): 639-656, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33896583

RESUMO

Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.


Assuntos
Sistemas CRISPR-Cas/genética , Reparo do DNA/genética , Edição de Genes/tendências , Genoma Humano/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga/genética , Humanos , Mutagênese Insercional/genética , Transdução de Sinais/genética
13.
Mol Cell ; 81(5): 1043-1057.e8, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33421364

RESUMO

Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.


Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Recombinação Homóloga , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Imagem Individual de Molécula
14.
Mol Cell ; 81(5): 1074-1083.e5, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33453169

RESUMO

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.


Assuntos
Proteína BRCA2/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Trifosfato de Adenosina/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo , Transdução de Sinais , Imagem Individual de Molécula
15.
Nucleic Acids Res ; 49(1): 285-305, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332547

RESUMO

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.


Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Proteínas Motores Moleculares/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/ultraestrutura , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Proteínas Motores Moleculares/ultraestrutura , Mutação de Sentido Incorreto , Mutação Puntual , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , RecQ Helicases/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por Substrato
17.
EMBO J ; 39(20): e105705, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32790929

RESUMO

Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.


Assuntos
Pareamento Cromossômico , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Topoisomerases/metabolismo , DNA de Cadeia Simples/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Domínio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , DNA Topoisomerases/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , Rad51 Recombinase/genética , Proteínas Recombinantes , Proteínas de Saccharomyces cerevisiae/genética
18.
J Vis Exp ; (160)2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32658186

RESUMO

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.


Assuntos
DNA/genética , Recombinação Homóloga , Dispositivos Lab-On-A-Chip , DNA/química , Humanos
19.
Cell ; 181(6): 1380-1394.e18, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32502392

RESUMO

Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.


Assuntos
Trifosfato de Adenosina/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , DNA/genética , Recombinação Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Hidrólise , Saccharomyces cerevisiae/genética , Alinhamento de Sequência/métodos
20.
Mol Cell ; 79(1): 99-114.e9, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32445620

RESUMO

Structural maintenance of chromosomes (SMC) complexes are essential for genome organization from bacteria to humans, but their mechanisms of action remain poorly understood. Here, we characterize human SMC complexes condensin I and II and unveil the architecture of the human condensin II complex, revealing two putative DNA-entrapment sites. Using single-molecule imaging, we demonstrate that both condensin I and II exhibit ATP-dependent motor activity and promote extensive and reversible compaction of double-stranded DNA. Nucleosomes are incorporated into DNA loops during compaction without being displaced from the DNA, indicating that condensin complexes can readily act upon nucleosome-bound DNA molecules. These observations shed light on critical processes involved in genome organization in human cells.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula/métodos
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