RESUMEN
Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.
Asunto(s)
Proteínas de Escherichia coli , Ácidos Nucleicos Heterodúplex , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genética , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , ADN/genética , ADN/metabolismo , Fenómenos MagnéticosRESUMEN
RNA-DNA hybrids are often associated with genome instability and also function as a cellular regulator in many biological processes. In this study, we show that accumulated RNA-DNA hybrids cause multiple defects in budding yeast meiosis, including decreased sporulation efficiency and spore viability. Further analysis shows that these RNA-DNA hybrid foci colocalize with RPA/Rad51 foci on chromosomes. The efficient formation of RNA-DNA hybrid foci depends on Rad52 and ssDNA ends of meiotic DNA double-strand breaks (DSBs), and their number is correlated with DSB frequency. Interestingly, RNA-DNA hybrid foci and recombination foci show similar dynamics. The excessive accumulation of RNA-DNA hybrids around DSBs competes with Rad51/Dmc1, impairs homolog bias, and decreases crossover and noncrossover recombination. Furthermore, precocious removal of RNA-DNA hybrids by RNase H1 overexpression also impairs meiotic recombination similarly. Taken together, our results demonstrate that RNA-DNA hybrids form at ssDNA ends of DSBs to actively regulate meiotic recombination.
Asunto(s)
ADN de Hongos/metabolismo , Recombinación Homóloga , Meiosis , Ácidos Nucleicos Heterodúplex/metabolismo , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , ADN de Hongos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , ARN de Hongos/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in â¼15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT.
Asunto(s)
ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/enzimología , Ácidos Nucleicos Heterodúplex/metabolismo , Homeostasis del Telómero , Telómero/metabolismo , Línea Celular Tumoral , Reparación de la Incompatibilidad de ADN , ADN de Neoplasias/genética , Proteínas de Unión al ADN/genética , Inestabilidad Genómica , Células HeLa , Humanos , Modelos Genéticos , Proteína 2 Homóloga a MutS/genética , Neoplasias/genética , Neoplasias/patología , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Telómero/genéticaRESUMEN
Repair of DNA double-stranded breaks by homologous recombination (HR) is dependent on DNA end resection and on post-translational modification of repair factors. In budding yeast, single-stranded DNA is coated by replication protein A (RPA) following DNA end resection, and DNA-RPA complexes are then SUMO-modified by the E3 ligase Siz2 to promote repair. Here, we show using enzymatic assays that DNA duplexes containing 3' single-stranded DNA overhangs increase the rate of RPA SUMO modification by Siz2. The SAP domain of Siz2 binds DNA duplexes and makes a key contribution to this process as highlighted by models and a crystal structure of Siz2 and by assays performed using protein mutants. Enzymatic assays performed using DNA that can accommodate multiple RPA proteins suggest a model in which the SUMO-RPA signal is amplified by successive rounds of Siz2-dependent SUMO modification of RPA and dissociation of SUMO-RPA at the junction between single- and double-stranded DNA. Our results provide insights on how DNA architecture scaffolds a substrate and E3 ligase to promote SUMO modification in the context of DNA repair.
Asunto(s)
Ácidos Nucleicos Heterodúplex/metabolismo , Proteína de Replicación A/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Cristalografía por Rayos X , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Polarización de Fluorescencia , Mutación , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Dominios Proteicos , Proteína de Replicación A/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Contractile forces within the planar interface between T cell and antigen-presenting surface mechanically stimulate T cell receptors (TCR) in the mature immune synapses. However, the origin of mechanical stimulation during the initial, i.e., presynaptic, microvilli-based TCR activation in the course of immune surveillance remains unknown and new tools to help address this problem are needed. In this work, we develop nucleic acid nanoassembly (NAN)-based technology for functionalization of hydrogels using isothermal toehold-mediated reassociation of RNA/DNA heteroduplexes. Resulting platform allows for regulation with NAN linkers of 3D force momentum along the TCR mechanical axis, whereas hydrogels contribute to modulation of 2D shear modulus. By utilizing different lengths of NAN linkers conjugated to polyacrylamide gels of different shear moduli, we demonstrate an efficient capture of human T lymphocytes and tunable activation of TCR, as confirmed by T-cell spreading and pY foci.
Asunto(s)
Hidrogeles/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/genética , Células Presentadoras de Antígenos/efectos de los fármacos , ADN/química , ADN/farmacología , Humanos , Hidrogeles/química , Activación de Linfocitos/genética , Linfocitos/metabolismo , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/farmacología , ARN/química , ARN/genética , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
Eukaryotic genomes contain many duplicated genes closely located with each other, such as the hexose transporter (HXT) genes in Saccharomyces cerevisiae. They can potentially recombine via single-strand annealing (SSA) pathway. SSA between highly divergent sequences generates heteroduplex DNA intermediates with many mismatches, which can be corrected by mismatch repair (MMR), resulting in recombinant sequences with a single junction point. In this report, we demonstrate that SSA between HXT1 and HXT4 genes in MMR-deficient yeast cells produces recombinant genes with multiple-junctions resulting from alternating HXT1 and HXT4 tracts. The mutations in MMR genes had differential effects on SSA frequencies; msh6Δ mutation significantly stimulated SSA events, whereas msh2Δ and msh3Δ slightly suppressed it. We set up an assay that can identify a pair of recombinant genes derived from a single heteroduplex DNA. As a result, the recombinant genes with multiple-junctions were found to accompany genes with single-junctions. Based on the results presented here, a model was proposed to generate multiple-junctions in SSA pathway involving an alternative short-patch repair system.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Proteínas de Transporte de Monosacáridos/genética , Ácidos Nucleicos Heterodúplex/genética , Saccharomyces cerevisiae/genética , Disparidad de Par Base , ADN de Hongos , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mutación , Recombinación GenéticaRESUMEN
Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.
Asunto(s)
Proteínas de Homeodominio/química , Ácidos Nucleicos Heterodúplex/química , Secuencias de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Dominios Proteicos , Relación Estructura-Actividad , Recombinación V(D)JRESUMEN
During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.
Asunto(s)
ADN/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión/genética , ADN/genética , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/genética , Reparación del ADN/fisiología , ADN de Cadena Simple/genética , Recombinación Homóloga/genética , Recombinación Homóloga/fisiología , Humanos , Mutación/genética , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Estructura Secundaria de Proteína , Recombinasa Rad51/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genéticaRESUMEN
In this study, we demonstrate that 5S ribosomal RNA (rRNA), a highly structured and protein-bound RNA, is quite difficult to reduce with antisense oligonucleotides (ASOs). However, we found a single accessible site that was targetable with a high-affinity complementary ASO. The ASO appeared to bind to the site, recruit RNaseH1, and cause degradation of the 5S RNA. Intriguingly, we also observed that the same ASO induced an accumulation of pre-5S RNA, which may contribute to reduced levels of mature 5S rRNA. As expected, ASO mediated reduction of 5S RNA, and modest inhibition of processing of pre-5S RNA resulted in nucleolar toxicity. However, the toxicity induced was minimal compared with actinomycin D, consistent with its modest effects on pre-5S rRNA. Mechanistically, we show that the accumulation of pre-5S rRNA required ASO hybridization to the cognate rRNA sequence but was independent of RNaseH1 activity. We found that Ro60 and La, proteins known to bind misprocessed RNAs, likely sequester the ASO-pre-5S rRNA species and block RNaseH1 activity, thus identifying another example of competitive mechanisms mediated by proteins that compete with RNaseH1 for binding to ASO-RNA heteroduplexes.
Asunto(s)
Ácidos Nucleicos Heterodúplex/genética , Oligonucleótidos Antisentido/genética , ARN Mensajero/genética , ARN Ribosómico 5S/genética , Humanos , Ácidos Nucleicos Heterodúplex/farmacología , Oligonucleótidos Antisentido/farmacología , Unión Proteica/genética , Proteínas/genética , Estabilidad del ARN/efectos de los fármacos , ARN Ribosómico 5S/efectos de los fármacos , Ribonucleasa H/genéticaRESUMEN
Crossover recombination is critical for meiotic chromosome segregation, but how mammalian crossing over is accomplished is poorly understood. Here, we illuminate how strands exchange during meiotic recombination in male mice by analyzing patterns of heteroduplex DNA in recombinant molecules preserved by the mismatch correction deficiency of Msh2-/- mutants. Surprisingly, MSH2-dependent recombination suppression was not evident. However, a substantial fraction of crossover products retained heteroduplex DNA, and some provided evidence of MSH2-independent correction. Biased crossover resolution was observed, consistent with asymmetry between DNA ends in earlier intermediates. Many crossover products yielded no heteroduplex DNA, suggesting dismantling by D-loop migration. Unlike the complexity of crossovers in yeast, these simple modifications of the original double-strand break repair model-asymmetry in recombination intermediates and D-loop migration-may be sufficient to explain most meiotic crossing over in mice while also addressing long-standing questions related to Holliday junction resolution.
Asunto(s)
Intercambio Genético/fisiología , Recombinación Homóloga/fisiología , Meiosis/fisiología , Animales , Segregación Cromosómica/genética , Intercambio Genético/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Recombinación Homóloga/genética , Masculino , Meiosis/genética , Ratones , Ratones Endogámicos DBA , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ácidos Nucleicos Heterodúplex/genéticaRESUMEN
Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry. We have chosen AvaII (G↓GWCC, where W stands for A or T) as a representative of this group of restriction endonucleases for detailed characterization. Here, we report crystal structures of AvaII alone, in specific complex with partially cleaved dsDNA, and in scanning complex with an RNA/DNA hybrid. The specific complex reveals a novel form of semi-specific dsDNA readout by a hexa-coordinated metal cation, most likely Ca2+ or Mg2+. Substitutions of residues anchoring this non-catalytic metal ion severely impair DNA binding and cleavage. The dsDNA in the AvaII complex is in the A-like form. This creates space for 2'-OH groups to be accommodated without intra-nucleic acid steric conflicts. PD-(D/E)XK restriction endonucleases of known structure that bind their dsDNA targets in the A-like form cluster into structurally similar groups. Most such enzymes, including some not previously studied in this respect, cleave RNA/DNA heteroduplexes. We conclude that A-form dsDNA binding is a good predictor for RNA/DNA cleavage activity.
Asunto(s)
Enzimas de Restricción del ADN/ultraestructura , ADN/ultraestructura , Ácidos Nucleicos Heterodúplex/ultraestructura , ARN/ultraestructura , Anabaena variabilis/genética , Sitios de Unión/genética , Cristalografía por Rayos X , ADN/genética , Roturas del ADN de Doble Cadena , Enzimas de Restricción del ADN/genética , Metales/química , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , ARN/genéticaRESUMEN
In the last decade, it has become evident that RNA is frequently found in DNA. It is now well established that single embedded ribonucleoside monophosphates (rNMPs) are primarily introduced by DNA polymerases and that longer stretches of RNA can anneal to DNA, generating RNA:DNA hybrids. Among them, the most studied are R-loops, peculiar three-stranded nucleic acid structures formed upon the re-hybridization of a transcript to its template DNA. In addition, polyribonucleotide chains are synthesized to allow DNA replication priming, double-strand breaks repair, and may as well result from the direct incorporation of consecutive rNMPs by DNA polymerases. The bright side of RNA into DNA is that it contributes to regulating different physiological functions. The dark side, however, is that persistent RNA compromises genome integrity and genome stability. For these reasons, the characterization of all these structures has been under growing investigation. In this review, we discussed the origin of single and multiple ribonucleotides in the genome and in the DNA of organelles, focusing on situations where the aberrant processing of RNA:DNA hybrids may result in multiple rNMPs embedded in DNA. We concluded by providing an overview of the currently available strategies to study the presence of single and multiple ribonucleotides in DNA in vivo.
Asunto(s)
ADN/química , Inestabilidad Genómica , Ácidos Nucleicos Heterodúplex/química , Ribonucleótidos/química , Animales , ADN/genética , Replicación del ADN , Humanos , Ácidos Nucleicos Heterodúplex/genética , Estructuras R-Loop , Ribonucleótidos/genéticaRESUMEN
Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.
Asunto(s)
Adenosina Trifosfato/química , ADN de Cadena Simple/química , ADN/química , Recombinación Homóloga , Ácidos Nucleicos Heterodúplex/química , Rec A Recombinasas/química , Adenosina Trifosfato/metabolismo , Bacterias/genética , Bacterias/metabolismo , Sitios de Unión , ADN/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Hidrólisis , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Unión Proteica , Conformación Proteica , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismoRESUMEN
AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.
Asunto(s)
ADN de Cadena Simple/genética , Silenciador del Gen , MicroARNs/genética , Ácidos Nucleicos Heterodúplex/genética , Oligonucleótidos Antisentido/genética , Animales , Northern Blotting , ADN de Cadena Simple/metabolismo , Femenino , Regulación de la Expresión Génica , Riñón/metabolismo , Hígado/metabolismo , Ratones Endogámicos ICR , MicroARNs/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , Ácidos Nucleicos Heterodúplex/farmacocinética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/metabolismoRESUMEN
The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Ácidos Nucleicos Heterodúplex/genética , ARN Guía de Kinetoplastida/genética , Animales , Disparidad de Par Base/genética , Línea Celular , Edición Génica/normas , Genes erbB-1/genética , Ácidos Nucleicos Heterodúplex/química , ARN Guía de Kinetoplastida/química , PorcinosRESUMEN
The post-replicative mismatch repair (MMR) system has anti-recombination activity that limits interactions between diverged sequences by recognizing mismatches in strand-exchange intermediates. In contrast to their equivalent roles during replication-error repair, mismatch recognition is more important for anti-recombination than subsequent mismatch processing. To obtain insight into this difference, ectopic substrates with 2% sequence divergence were used to examine mitotic recombination outcome (crossover or noncrossover; CO and NCO, respectively) and to infer molecular intermediates formed during double-strand break repair in Saccharomyces cerevisiae. Experiments were performed in an MMR-proficient strain, a strain with compromised mismatch-recognition activity (msh6Δ) and a strain that retained mismatch-recognition activity but was unable to process mismatches (mlh1Δ). While the loss of either mismatch binding or processing elevated the NCO frequency to a similar extent, CO events increased only when mismatch binding was compromised. The molecular features of NCOs, however, were altered in fundamentally different ways depending on whether mismatch binding or processing was eliminated. These data suggest a model in which mismatch recognition reverses strand-exchange intermediates prior to the initiation of end extension, while subsequent mismatch processing that is linked to end extension specifically destroys NCO intermediates that contain conflicting strand-discrimination signals for mismatch removal.
Asunto(s)
Reparación de la Incompatibilidad de ADN/genética , Proteínas de Unión al ADN/genética , Mitosis/genética , Homólogo 1 de la Proteína MutL/genética , Recombinación Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Disparidad de Par Base/genética , Intercambio Genético/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Replicación del ADN/genética , Ácidos Nucleicos Heterodúplex/genética , Saccharomyces cerevisiae/genéticaRESUMEN
During transcription, the nascent RNA strand can base pair with its template DNA, displacing the non-template strand as ssDNA and forming a structure called an R-loop. R-loops are common across many domains of life and cause DNA damage in certain contexts. In this review, we summarize recent results implicating R-loops as important regulators of cellular processes such as transcription termination, gene regulation, and DNA repair. We also highlight recent work suggesting that R-loops can be problematic to cells as blocks to efficient transcription and replication that trigger the DNA damage response. Finally, we discuss how R-loops may contribute to cancer, neurodegeneration, and inflammatory diseases and compare the available next-generation sequencing-based approaches to map R-loops genome wide.
Asunto(s)
Núcleo Celular/fisiología , ADN/genética , Genoma , Inestabilidad Genómica , Ácidos Nucleicos Heterodúplex/genética , ARN/genética , Animales , ADN/química , ADN/metabolismo , Daño del ADN , Reparación del ADN , Regulación de la Expresión Génica , Humanos , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , ARN/química , ARN/metabolismo , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.
Asunto(s)
Daño del ADN , Replicación del ADN , Inestabilidad Genómica , Neoplasias/genética , División del ARN , Precursores del ARN/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Transporte Activo de Núcleo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Poliadenilación , Precursores del ARN/biosíntesis , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteínas de Unión al ARNRESUMEN
Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.
Asunto(s)
ADN/genética , Exodesoxirribonucleasa V/genética , Genoma Bacteriano/genética , Secuencia de Bases , ADN/biosíntesis , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Replicación del ADN/genética , ADN de Cadena Simple/genética , Escherichia coli/genética , Ácidos Nucleicos Heterodúplex/genética , Rec A Recombinasas/genética , Recombinación GenéticaRESUMEN
TILLING (Targeting Induced Local Lesions IN Genomes), a popular reverse genetics approach in barley research, combines plant mutagenesis with efficient mutation detection for studying biological function of a specific gene. The high mutation frequency within a TILLING population principally enables the identification of induced variations in (almost) all genes of a given species (more precisely a given genotype of a species) of interest, which can be tested for their functional impact on morphological and/or physiological characteristics of the plant. Several TILLING populations induced by chemical mutagenesis were established for barley (Talame et al., Plant Biotechnol J 6:477-485, 2008; Gottwald et al., BMC Res Notes 2:258, 2009; Caldwell et al. Plant J 40:143-150, 2004) and showed the possibility for adapting protocols to develop further populations. This chapter describes a chemical mutagenesis protocol for barley seeds and two independent procedures for efficient single nucleotide polymorphism (SNP) detection in a large number of mutagenized plants either by slab-gel- or capillary gel-based electrophoreses on the LI-COR 4300 DNA Analyzer and the AdvanCE FS96 instruments, respectively.