RESUMO
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
Assuntos
Trifosfato de Adenosina , DNA Helicases , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Imagem Individual de Molécula , Terminação da Transcrição Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Polimerase II/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Imagem Individual de Molécula/métodos , RNA Helicases/metabolismo , RNA Helicases/genética , Transcrição Gênica , RNA Fúngico/metabolismo , RNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/genética , HidróliseRESUMO
Sen1 is an essential helicase for factor-dependent transcription termination in Saccharomyces cerevisiae, whose molecular-motor mechanism has not been well addressed. Here, we use single-molecule experimentation to better understand the molecular-motor determinants of its action on RNA polymerase II (Pol II) complex. We quantify Sen1 translocation activity on single-stranded DNA (ssDNA), finding elevated translocation rates, high levels of processivity and ATP affinities. Upon deleting the N- and C-terminal domains, or further deleting different parts of the prong subdomain, which is an essential element for transcription termination, Sen1 displays changes in its translocation properties, such as slightly reduced translocation processivities, enhanced translocation rates and statistically identical ATP affinities. Although these parameters fulfil the requirements for Sen1 translocating along the RNA transcript to catch up with a stalled Pol II complex, we observe significant reductions in the termination efficiencies as well as the factions of the formation of the previously described topological intermediate prior to termination, suggesting that the prong may preserve an interaction with Pol II complex during factor-dependent termination. Our results underscore a more detailed rho-like mechanism of Sen1 and a critical interaction between Sen1 and Pol II complex for factor-dependent transcription termination in eukaryotes.
Assuntos
DNA Helicases , RNA Helicases , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina , DNA de Cadeia Simples/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição Genética , DNA Helicases/metabolismoRESUMO
Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , Genoma , Streptococcus pyogenes/metabolismo , Edição de GenesRESUMO
Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 106 seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors.
Assuntos
DNA Super-Helicoidal , Proteínas de Escherichia coli/metabolismo , Escherichia coli , DNA Super-Helicoidal/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA/genética , RNA/metabolismo , Transcrição GênicaRESUMO
R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.
Assuntos
Proteínas de Bactérias/metabolismo , Estruturas R-Loop , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Mutação , Imagem Individual de Molécula , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a â¼2 s synapsis between blunt DNA ends that is increased to â¼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , DNA/metabolismo , DNA Ligases/metabolismo , Autoantígeno Ku/química , Multimerização ProteicaRESUMO
Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Ligação Proteica , Multimerização ProteicaRESUMO
Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds â¼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds â¼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.
Assuntos
DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases/metabolismo , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Thermus/genéticaRESUMO
Over the past few decades, single-molecule manipulation has been widely applied to the real-time analysis of biomolecular interactions. It has enabled researchers to decipher structure-function relationships for polymers, enzymes, and larger-scale molecular machines, in particular by harnessing force to probe both chemical and mechanical stabilities. Nucleic acids have played a central role in this effort because, in addition to their biological significance, they exhibit unique polymeric properties which have recast them as key components participating in numerous experimental designs. In this review, we introduce recent developments highlighting this dual nature of nucleic acids in biophysics, as objects of study but also as tools allowing novel approaches. More specifically, we present molecular scaffolds as an emerging concept and describe their use in single-molecule force spectroscopy. Aspects related to folding and noncovalent interactions will be presented in parallel to research in enzymology, with a focus on the acquisition of thermodynamic and kinetic data.
Assuntos
DNA , Imagem Individual de Molécula , Pareamento de Bases , Fenômenos Biomecânicos , DNA/química , DNA/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.
Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Bacteriano/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas MutL/metabolismo , Pareamento Incorreto de Bases/genética , Proteína 9 Associada à CRISPR/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Endodesoxirribonucleases/metabolismo , Instabilidade Genômica/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismoRESUMO
The residence time of a drug on its target has been suggested as a more pertinent metric of therapeutic efficacy than the traditionally used affinity constant. Here, we introduce junctured-DNA tweezers as a generic platform that enables real-time observation, at the single-molecule level, of biomolecular interactions. This tool corresponds to a double-strand DNA scaffold that can be nanomanipulated and on which proteins of interest can be engrafted thanks to widely used genetic tagging strategies. Thus, junctured-DNA tweezers allow a straightforward and robust access to single-molecule force spectroscopy in drug discovery, and more generally in biophysics. Proof-of-principle experiments are provided for the rapamycin-mediated association between FKBP12 and FRB, a system relevant in both medicine and chemical biology. Individual interactions were monitored under a range of applied forces and temperatures, yielding after analysis the characteristic features of the energy profile along the dissociation landscape.
Assuntos
DNA/química , Nanoestruturas/química , Mapeamento de Interação de Proteínas/métodos , Animais , DNA de Cadeia Simples/química , Humanos , Modelos Moleculares , Nanotecnologia/métodos , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismoRESUMO
Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
RESUMO
All active living organisms mitigate DNA damage via DNA repair, and the so-called nucleotide excision repair pathway represents a functionally major part of the cell's DNA repair repertoire [1]. In this pathway, the damaged strand of DNA is incised and removed before being resynthesized. This form of DNA repair requires a multitude of proteins working in a complex choreography. Repair thus typically involves detection of a DNA lesion, validation of that detection event, search for an appropriate incision site and subsequent DNA incision, DNA unwinding/removal, and DNA resynthesis and religation. These activities are ultimately the result of molecules randomly diffusing and bumping into each other and acting in succession. It is also true, however, that repair components are often assembled into functional complexes which may be more efficient or regular in their mode of action. Studying DNA repair complexes for their mechanisms of assembly, action, and disassembly can help address fundamental questions such as whether DNA repair pathways are branched or linear; whether, for instance, they tolerate fluctuations in numbers of components; and more broadly how search processes between macromolecules take place or can be enhanced.
Assuntos
Reparo do DNA , Complexos Multiproteicos/metabolismo , Transcrição Gênica , Animais , Dano ao DNA , Humanos , Modelos Moleculares , Transporte Proteico , Imagem Individual de MoléculaRESUMO
Repairing DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nanomanipulation, allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and the addition of PAXX extends this lifetime to ~2 s. Further addition of XRCC4, XLF and ligase IV results in minute-scale synapsis and leads to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kilocalories per mole. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Técnicas Genéticas/instrumentação , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Pareamento Cromossômico , DNA/genética , DNA/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Fosforilação , Células Sf9 , SpodopteraRESUMO
DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.
Assuntos
Proteínas Arqueais/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Catenado/metabolismo , Sulfolobus solfataricus/enzimologia , Proteínas Arqueais/genética , Sequência de Bases , DNA Topoisomerases Tipo I/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA Catenado/química , DNA Catenado/genética , DNA Concatenado/química , DNA Concatenado/genética , DNA Concatenado/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Temperatura Alta , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Sulfolobus solfataricus/genéticaRESUMO
During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded 'transcription bubble,' and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA 'scrunching' and 'anti-scrunching,' the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.