RESUMEN

The demand for direct observation of biomolecular interactions provides new insights into the molecular mechanisms underlying many biological processes. Single-molecule imaging techniques enable real-time visualization of individual biomolecules, providing direct observations of protein machines. Various single-molecule imaging techniques have been developed and have contributed to breakthroughs in biological research. One such technique is the DNA curtain, a novel, high-throughput, single-molecule platform that integrates lipid fluidity, nano-fabrication, microfluidics, and fluorescence imaging. Many DNA metabolic reactions, such as replication, transcription, and chromatin dynamics, have been studied using DNA curtains. In particular, the DNA curtain platform has been intensively applied in investigating the molecular details of DNA repair processes. This article reviews DNA curtain techniques and their applications for imaging DNA repair proteins.

Asunto(s)

Reparación del ADN , ADN , ADN/metabolismo , Cromatina , Nanotecnología/métodos

RESUMEN

R-loops are nucleic acid structures containing a DNA-RNA hybrid and the associated non-template single-stranded DNA. R-loops are not only involved in many biological processes but also cause genomic instability when they are abnormally regulated. The R-loop regulation pathway consists of multiple steps associated with diverse proteins. The initial and essential step of the pathway is to recognize R-loops in long DNA of human genome. To elucidate the molecular mechanism underlying R-loop recognition by proteins, we utilize a novel high-throughput single-molecule approach called "DNA curtain" as well as electrophoretic mobility shift assays. Here, we describe the detailed protocols for these techniques that both can be used for studying the R-loop recognition mechanisms.

Asunto(s)

ADN , Estructuras R-Loop , ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Inestabilidad Genómica , Humanos , ARN/genética

RESUMEN

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.

RESUMEN

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.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Designer virus-inspired proteins drive the manufacturing of more effective, safer gene-delivery systems and simpler models to study viral assembly. However, self-assembly of engineered viromimetic proteins on specific nucleic acid templates, a distinctive viral property, has proved difficult. Inspired by viral packaging signals, we harness the programmability of CRISPR-Cas12a to direct the nucleation and growth of a self-assembling synthetic polypeptide into virus-like particles (VLP) on specific DNA molecules. Positioning up to ten nuclease-dead Cas12a (dCas12a) proteins along a 48.5 kbp DNA template triggers particle growth and full DNA encapsidation at limiting polypeptide concentrations. Particle growth rate is further increased when dCas12a is dimerized with a polymerization silk-like domain. Such improved self-assembly efficiency allows for discrimination between cognate versus noncognate DNA templates by the synthetic polypeptide. CRISPR-guided VLPs will help to develop programmable bioinspired nanomaterials with applications in biotechnology as well as viromimetic scaffolds to improve our understanding of viral self-assembly.

Asunto(s)

Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Virión , ADN , Nucleocápside , Ensamble de Virus/genética

RESUMEN

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1- DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

Asunto(s)

ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Histonas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Imagen Individual de Molécula , ADN de Hongos/metabolismo , Fotoblanqueo , Unión Proteica , Multimerización de Proteína

RESUMEN

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.

Asunto(s)

Proteína BRCA2/genética , Replicación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/genética , Recombinasa Rad51/genética , Adenosina Trifosfato/metabolismo , Proteína BRCA2/metabolismo , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hidrólisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Unión al ARN/metabolismo , Recombinasa Rad51/metabolismo , Transducción de Señal , Imagen Individual de Molécula , Proteína Fluorescente Roja

RESUMEN

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.

Asunto(s)

Adenosina Trifosfato/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , ADN/genética , Recombinación Homóloga/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Hidrólisis , Saccharomyces cerevisiae/genética , Alineación de Secuencia/métodos

RESUMEN

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.

Asunto(s)

Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Nucleosomas/metabolismo , Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Complejos Multiproteicos/genética , Unión Proteica , Conformación Proteica , Imagen Individual de Molécula/métodos

RESUMEN

DNA curtain is a high-throughput system, integrating a lipid bilayer, fluorescence imaging, and microfluidics to probe protein-DNA interactions in real-time and has provided in-depth understanding of DNA metabolism. Especially, the microfluidic platform of a DNA curtain is highly suitable for a biochip. In the DNA curtain, DNA molecules are aligned along chromium nanobarriers, which are fabricated on a slide surface, and visualized using an intercalating dye, YOYO-1. Although the chromium barriers confer precise geometric alignment of DNA, reuse of the slides is limited by wear of the barriers during cleaning. YOYO-1 is rapidly photobleached and causes photocleavage of DNA under continuous laser illumination, restricting DNA observation to a brief time window. To address these challenges, we developed a new nanopatterned slide, upon which carved nanotrenches serve as diffusion barriers. The nanotrenches were robust under harsh cleaning conditions, facilitating the maintenance of surface cleanliness that is essential to slide reuse. We also stained DNA with a fluorescent protein with a DNA-binding motif, fluorescent protein-DNA binding peptide (FP-DBP). FP-DBP was slowly photobleached and did not cause DNA photocleavage. This new DNA curtain system enables a more stable and repeatable investigation of real-time protein-DNA interactions and will serve as a good platform for lab-on-a-chip.

Asunto(s)

Benzoxazoles/análisis , Proteínas de Unión al ADN/análisis , ADN/análisis , Colorantes Fluorescentes/análisis , Nanoestructuras/química , Compuestos de Quinolinio/análisis , Imagen Individual de Molécula/métodos , Membrana Dobles de Lípidos/química , Imagen Óptica/métodos

RESUMEN

Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein-protein and protein-DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein-protein and protein-DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination.

Asunto(s)

Recombinación Homóloga/genética , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodos , Animales , ADN/genética , ADN Cruciforme/metabolismo , Humanos , Unión Proteica

RESUMEN

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Adenosina Trifosfato/metabolismo , Adenilil Imidodifosfato/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/genética , Hidrólisis , Proteínas Hierro-Azufre/química , Modelos Moleculares , Mutación , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Ácidos Nucleicos Heterodúplex , Dominios Proteicos , Imagen Individual de Molécula

RESUMEN

Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

Asunto(s)

ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Recombinación Homóloga , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ADN Helicasas/química , Proteínas Fluorescentes Verdes/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Eliminación de Secuencia

RESUMEN

In physiological settings, all nucleic acids motor proteins must travel along substrates that are crowded with other proteins. However, the physical basis for how motor proteins behave in these highly crowded environments remains unknown. Here, we use real-time single-molecule imaging to determine how the ATP-dependent translocase RecBCD travels along DNA occupied by tandem arrays of high-affinity DNA binding proteins. We show that RecBCD forces each protein into its nearest adjacent neighbor, causing rapid disruption of the protein-nucleic acid interaction. This mechanism is not the same way that RecBCD disrupts isolated nucleoprotein complexes on otherwise naked DNA. Instead, molecular crowding itself completely alters the mechanism by which RecBCD removes tightly bound protein obstacles from DNA.

Asunto(s)

Replicación del ADN/fisiología , ADN/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Exodesoxirribonucleasa V/metabolismo , Nucleoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Microscopía Fluorescente , Método de Montecarlo