RESUMEN
Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.
Asunto(s)
Escherichia coli , Edición de ARN , ARN sin Sentido , ARN Mensajero , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adenosina Desaminasa/metabolismo , Adenosina Desaminasa/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genéticaRESUMEN
CRISPR mutagenesis screens conducted with SpCas9 and other nucleases have identified certain cis-regulatory elements and genetic variants but at a limited resolution due to the absence of protospacer adjacent motif (PAM) sequences. Here, leveraging the broad targeting scope of the near-PAMless SpRY variant, we have demonstrated that saturated SpRY mutagenesis and base editing screens can faithfully identify functional regulatory elements and essential genetic variants for target gene expression at single-base resolution. We further extended this methodology to investigate a genome-wide association study (GWAS) locus at 10q22.1 associated with a red blood cell trait, where we identified potential enhancers regulating HK1 gene expression, despite not all of these enhancers exhibiting typical chromatin signatures. More importantly, our saturated base editing screens pinpoint multiple causal variants within this locus that would otherwise be missed by Bayesian statistical fine-mapping. Our approach is generally applicable to functional interrogation of all non-coding genomic elements while complementing other high-coverage CRISPR screens.
Asunto(s)
Sistemas CRISPR-Cas , Estudio de Asociación del Genoma Completo , Humanos , Estudio de Asociación del Genoma Completo/métodos , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Mutagénesis , Elementos de Facilitación Genéticos/genéticaRESUMEN
Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targeting to PAM-containing regions. Protein engineering has led to PAM-relaxed SpCas9 variants like SpG and SpRY. Given the evolved role of PAMs in facilitating target-searching kinetics, it remains unclear how these variants quickly locate their targets. We show that SpG and SpRY exhibit a preference for the seed region. To compensate for the relaxed PAM recognition, SpRY has evolved a sequence preference for the seed region through interactions with A61R and A1322R. Furthermore, SpCas9 exhibits a significant decrease in target search kinetics on high-PAM-density DNA, slowing down up to three orders of magnitude compared to low-PAM-density DNA, suggesting the necessity for sequence recognition even in PAM-relaxed variants. This underscores the importance of considering Cas9 target-searching kinetics in SpCas9 PAMless engineering, providing valuable insights for further PAMless Cas9 protein engineering efforts.
Asunto(s)
Proteína 9 Asociada a CRISPR , Humanos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , ADN/metabolismo , ADN/genética , Cinética , Edición Génica/métodos , Secuencia de Bases , Células HEK293RESUMEN
Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.
RESUMEN
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.
Asunto(s)
ADN de Cadena Simple , RecQ Helicasas , ADN/genética , ADN de Cadena Simple/genética , Recombinación Homóloga/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismoRESUMEN
Stoichiometric genome-scale metabolic network models (GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric ratios, other constraints such as enzyme availability and thermodynamic feasibility can also limit the phenotype solution space. Extended GEM models considering either enzymatic or thermodynamic constraints have been shown to improve prediction accuracy. In this paper, we propose a novel method that integrates both enzymatic and thermodynamic constraints in a single Pyomo modeling framework (ETGEMs). We applied this method to construct the EcoETM (E. coli metabolic model with enzymatic and thermodynamic constraints). Using this model, we calculated the optimal pathways for cellular growth and the production of 22 metabolites. When comparing the results with those of iML1515 and models with one of the two constraints, we observed that many thermodynamically unfavorable and/or high enzyme cost pathways were excluded from EcoETM. For example, the synthesis pathway of carbamoyl-phosphate (Cbp) from iML1515 is both thermodynamically unfavorable and enzymatically costly. After introducing the new constraints, the production pathways and yields of several Cbp-derived products (e.g. L-arginine, orotate) calculated using EcoETM were more realistic. The results of this study demonstrate the great application potential of metabolic models with multiple constraints for pathway analysis and phenotype prediction.
Asunto(s)
Escherichia coli , Modelos Biológicos , Escherichia coli/genética , Genoma Bacteriano/genética , Redes y Vías Metabólicas/genética , TermodinámicaRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas/genética , Reparación del ADN/genética , Edición Génica/tendencias , Genoma Humano/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Recombinación Homóloga/genética , Humanos , Mutagénesis Insercional/genética , Transducción de Señal/genéticaRESUMEN
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.
Asunto(s)
ADN Helicasas/metabolismo , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Roturas del ADN de Doble Cadena , ADN Helicasas/química , ADN Helicasas/genética , Reparación del ADN , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Hidrólisis , Mutación , Mycobacterium/enzimología , Mycobacterium/genética , Unión Proteica , Dominios ProteicosRESUMEN
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 RojaRESUMEN
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 â¼50%. 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.
Asunto(s)
ADN de Cadena Simple/metabolismo , Recombinación Homóloga , Proteínas Motoras Moleculares/metabolismo , RecQ Helicasas/metabolismo , Imagen Individual de Molécula , Adenosina Trifosfato/metabolismo , ADN de Cadena Simple/ultraestructura , Humanos , Hidrólisis , Cinética , Microscopía de Fuerza Atómica , Proteínas Motoras Moleculares/ultraestructura , Mutación Missense , Mutación Puntual , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/ultraestructura , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicación A/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
ADN/genética , Recombinación Homóloga , Dispositivos Laboratorio en un Chip , ADN/química , HumanosRESUMEN
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/métodosRESUMEN
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 MoléculaRESUMEN
Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (â¼70-80 base pairs per second) unwinding extensive tracts (â¼8-10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.
Asunto(s)
ADN de Cadena Simple/metabolismo , ADN/metabolismo , RecQ Helicasas/análisis , RecQ Helicasas/metabolismo , Imagen Individual de Molécula , Emparejamiento Base , Síndrome de Bloom/genética , ADN/química , Reparación del ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/química , Recombinación Homóloga , Humanos , Modelos Moleculares , Recombinasa Rad51/metabolismo , RecQ Helicasas/química , RecQ Helicasas/genética , Proteína de Replicación A/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Rad52 is a key factor for homologous recombination (HR) in yeast. Rad52 helps assemble Rad51-ssDNA nucleoprotein filaments that catalyze DNA strand exchange, and it mediates single-strand DNA annealing. We find that Rad52 has an even earlier function in HR in restricting DNA double-stranded break ends resection that generates 3' single-stranded DNA (ssDNA) tails. In fission yeast, Exo1 is the primary resection nuclease, with the helicase Rqh1 playing a minor role. We demonstrate that the choice of two extensive resection pathways is regulated by Rad52. In rad52 cells, the resection rate increases from â¼3-5 kb/h up to â¼10-20 kb/h in an Rqh1-dependent manner, while Exo1 becomes dispensable. Budding yeast Rad52 similarly inhibits Sgs1-dependent resection. Single-molecule analysis with purified budding yeast proteins shows that Rad52 competes with Sgs1 for DNA end binding and inhibits Sgs1 translocation along DNA. These results identify a role for Rad52 in limiting ssDNA generated by end resection.
Asunto(s)
Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Regulación Fúngica de la Expresión Génica , Cinética , Mutación , Dominios Proteicos , Transporte de Proteínas , Proteína Recombinante y Reparadora de ADN Rad52/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinación Homóloga , Nucleosomas/metabolismo , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
DNA helicases of the RecQ family are conserved among the three domains of life and play essential roles in genome maintenance. Mutations in several human RecQ helicases lead to diseases that are marked by cancer predisposition. The Saccharomyces cerevisiae RecQ helicase Sgs1 is orthologous to human BLM, defects in which cause the cancer-prone Bloom's Syndrome. Here, we use single-molecule imaging to provide a quantitative mechanistic understanding of Sgs1 activities on single stranded DNA (ssDNA), which is a central intermediate in all aspects of DNA metabolism. We show that Sgs1 acts upon ssDNA bound by either replication protein A (RPA) or the recombinase Rad51. Surprisingly, we find that Sgs1 utilizes a novel motor mechanism for disrupting ssDNA intermediates bound by the recombinase protein Rad51. The ability of Sgs1 to disrupt Rad51-ssDNA filaments may explain some of the defects engendered by RECQ helicase deficiencies in human cells.
Asunto(s)
Recombinasa Rad51/genética , RecQ Helicasas/genética , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfato/genética , Síndrome de Bloom/genética , Síndrome de Bloom/patología , Reparación del ADN/genética , ADN de Cadena Simple , Humanos , Mutación/genética , Saccharomyces cerevisiae/genéticaRESUMEN
CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative Enterobacteriaceae family, especially Escherichia coli and Pectobacterium atrosepticum, have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the Enterobacteriaceae. We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.
Asunto(s)
Bacteriófagos/fisiología , Sistemas CRISPR-Cas , Enterobacteriaceae/genética , Archaea/genética , Bacteriófagos/genética , Enterobacteriaceae/inmunología , Enterobacteriaceae/virología , Escherichia coli/genética , Interacciones Microbiota-Huesped , Pectobacterium/genéticaRESUMEN
Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Given the importance of Cas effectors in host defense and for biotechnology tools, there has been considerable interest in developing sensitive methods for detecting Cas effector activity through CRISPR interference. In this chapter, we describe an in vivo fluorescence-based method for monitoring plasmid interference in Escherichia coli. This approach uses a green fluorescent protein reporter to monitor varying plasmid levels within bacterial colonies, or to measure the rate of plasmid-loss in bacterial populations over time. We demonstrate the use of this simple plasmid-loss assay for both chromosomally integrated and plasmid-borne CRISPR-Cas systems.