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1.
Nucleic Acids Res ; 49(11): 6114-6127, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34125895

RESUMO

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as 'heterochiral' strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction - strand displacement from PNA-DNA heteroduplexes - remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA-DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA-DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.


Assuntos
DNA/química , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Peptídicos/química , Cinética , RNA/química , Estereoisomerismo , Termodinâmica
2.
Nat Methods ; 18(7): 816-820, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34127856

RESUMO

Single-molecule Förster resonance energy transfer (smFRET) has become a versatile and widespread method to probe nanoscale conformation and dynamics. However, current experimental modalities often resort to molecule immobilization for long observation times and do not always approach the resolution limit of FRET-based nanoscale metrology. Here we present ABEL-FRET, an immobilization-free platform for smFRET measurements with ultrahigh resolving power in FRET efficiency. Importantly, single-molecule diffusivity is used to provide additional size and shape information for hydrodynamic profiling of individual molecules, which, together with the concurrently measured intramolecular conformation through FRET, enables a holistic and dynamic view of biomolecules and their complexes.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Dano ao DNA , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Hidrodinâmica , Dispositivos Lab-On-A-Chip , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Fótons , Imagem Individual de Molécula/instrumentação
3.
Nucleic Acids Res ; 49(12): 6638-6659, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-33978760

RESUMO

G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date.


Assuntos
Quadruplex G , Oligonucleotídeos/química , Genoma Humano , Humanos , Ligantes , Ácidos Nucleicos Heteroduplexes , Ácidos Nucleicos Peptídicos , Transcriptoma
4.
J Cell Biol ; 220(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33830170

RESUMO

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.


Assuntos
Anticorpos Monoclonais/metabolismo , DNA/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Artefatos , DNA/química , Humanos , Ácidos Nucleicos Heteroduplexes/química , RNA/química , Ribonuclease H/química
5.
Yi Chuan ; 43(2): 182-193, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33724220

RESUMO

Homologous recombination is an important source of biological genetic variation. Limited by detection methods, there are only a few reports on the homologous recombination in high plants and its product - heteroduplex DNA (hDNA). In the present study, applying the strategy of detecting hDNA by constructing populations from inhibited post-meiotic segregation, two hybrid triploid populations were constructed from two maternal parents inPopulus tomentosa by inhibiting post-meiotic segregation. One hundred and ten simple sequence repeat (SSR) markers were used to study the occurrence and variation of hDNA on nine chromosomes inP. tomentosa with different genotypes. The results showed that the frequencies of hDNA between two female parents inP. tomentosa ranged from 8.5% to 87.2%. The hDNA frequency was positively correlated to the distance from the centromere, but the average hDNA frequency on a chromosome had no correlation with the chromosome length. One to 3 times recombination events were detected on most chromosomes, and only a few four- or five-times recombination events were detected. The overall frequencies of hDNA on the same chromosome in two genotypic individuals were roughly similar, while the hDNA frequencies varied greatly at specific SSR loci. Compared withTacamahaca poplar hybrid,P. pseudo-simonii × P. nigra 'Zheyin3#', detection of homologous recombination times and the frequency and location of hDNA were largely different. This study is the first to describe the characteristics and variations of homologous recombination inP. tomentosa with two different genotypes, which will provide valuable insights for exploring the characteristics and variations of homologous recombination among interspecies and intraspecies in higher plant.


Assuntos
Populus , Feminino , Genótipo , Recombinação Homóloga , Humanos , Repetições de Microssatélites/genética , Ácidos Nucleicos Heteroduplexes , Populus/genética
6.
Nucleic Acids Res ; 49(7): 4120-4128, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33764415

RESUMO

Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR , Endodesoxirribonucleases/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/farmacocinética , Edição de Genes , Modelos Moleculares , Ligação Proteica
7.
J Microbiol ; 59(4): 401-409, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33779953

RESUMO

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.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Transporte de Monossacarídeos/genética , Ácidos Nucleicos Heteroduplexes/genética , Saccharomyces cerevisiae/genética , Pareamento Incorreto de Bases , DNA Fúngico , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Mutação , Recombinação Genética
8.
J Vis Exp ; (167)2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33554969

RESUMO

The three-stranded nucleic acid structure, R-loop, is increasingly recognized for its role in gene regulation. Initially, R-loops were thought to be the by-products of transcription; but recent findings of fewer R-loops in diseased cells made it clear that R-loops have functional roles in a variety of human cells. Next, it is critical to understand the roles of R-loops and how cells balance their abundance. A challenge in the field is the quantitation of R-loops since much of the work relies on the S9.6 monoclonal antibody whose specificity for RNA-DNA hybrids has been questioned. Here, we use dot-blots with the S9.6 antibody to quantify R-loops and show the sensitivity and specificity of this assay with RNase H, RNase T1, and RNase III that cleave RNA-DNA hybrids, single-stranded RNA, and double-stranded RNA, respectively. This method is highly reproducible, uses general laboratory equipment and reagents, and provides results within two days. This assay can be used in research and clinical settings to quantify R-loops and assess the effect of mutations in genes such as senataxin on R-loop abundance.


Assuntos
Immunoblotting , Estruturas R-Loop , Anticorpos/metabolismo , DNA/isolamento & purificação , Fibroblastos/metabolismo , Humanos , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/metabolismo , Estruturas R-Loop/genética , RNA/genética , Ribonuclease H/metabolismo , Ribonucleases/metabolismo
9.
EMBO J ; 40(7): e106018, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33634895

RESUMO

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.


Assuntos
Proteína BRCA2/metabolismo , RNA Helicases DEAD-box/metabolismo , Reparo de DNA por Recombinação , Proteína BRCA2/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Humanos , Ácidos Nucleicos Heteroduplexes , Ligação Proteica
10.
Methods Mol Biol ; 2153: 535-554, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840803

RESUMO

DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIR in various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defects for genomic instability by MIR.


Assuntos
DNA Fúngico/genética , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , DNA Fúngico/química , Proteínas de Escherichia coli/metabolismo , Mutação , Ácidos Nucleicos Heteroduplexes , Saccharomyces cerevisiae/metabolismo
11.
Nature ; 587(7833): 291-296, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33087930

RESUMO

Transcription factors recognize specific genomic sequences to regulate complex gene-expression programs. Although it is well-established that transcription factors bind to specific DNA sequences using a combination of base readout and shape recognition, some fundamental aspects of protein-DNA binding remain poorly understood1,2. Many DNA-binding proteins induce changes in the structure of the DNA outside the intrinsic B-DNA envelope. However, how the energetic cost that is associated with distorting the DNA contributes to recognition has proven difficult to study, because the distorted DNA exists in low abundance in the unbound ensemble3-9. Here we use a high-throughput assay that we term SaMBA (saturation mismatch-binding assay) to investigate the role of DNA conformational penalties in transcription factor-DNA recognition. In SaMBA, mismatched base pairs are introduced to pre-induce structural distortions in the DNA that are much larger than those induced by changes in the Watson-Crick sequence. Notably, approximately 10% of mismatches increased transcription factor binding, and for each of the 22 transcription factors that were examined, at least one mismatch was found that increased the binding affinity. Mismatches also converted non-specific sites into high-affinity sites, and high-affinity sites into 'super sites' that exhibit stronger affinity than any known canonical binding site. Determination of high-resolution X-ray structures, combined with nuclear magnetic resonance measurements and structural analyses, showed that many of the DNA mismatches that increase binding induce distortions that are similar to those induced by protein binding-thus prepaying some of the energetic cost incurred from deforming the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit transcription factors and thus modulate replication and repair activities in the cell10,11.


Assuntos
Proteínas de Ligação a DNA/química , Conformação Molecular , Ácidos Nucleicos Heteroduplexes/química , Proteínas de Arabidopsis/química , Pareamento de Bases , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica , Fatores de Transcrição/química
12.
Int J Mol Sci ; 21(20)2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33096752

RESUMO

Certain G-quadruplex forming guanine-rich oligonucleotides (GROs), including AS1411, are endowed with cancer-selective antiproliferative activity. They are known to bind to nucleolin protein, resulting in the inhibition of nucleolin-mediated phenomena. However, multiple nucleolin-independent biological effects of GROs have also been reported, allowing them to be considered promising candidates for multi-targeted cancer therapy. Herein, with the aim of optimizing AS1411 structural features to find GROs with improved anticancer properties, we have studied a small library of AS1411 derivatives differing in the sequence length and base composition. The AS1411 derivatives were characterized by using circular dichroism and nuclear magnetic resonance spectroscopies and then investigated for their enzymatic resistance in serum and nuclear extract, as well as for their ability to bind nucleolin, inhibit topoisomerase I, and affect the viability of MCF-7 human breast adenocarcinoma cells. All derivatives showed higher thermal stability and inhibitory effect against topoisomerase I than AS1411. In addition, most of them showed an improved antiproliferative activity on MCF-7 cells compared to AS1411 despite a weaker binding to nucleolin. Our results support the hypothesis that the antiproliferative properties of GROs are due to multi-targeted effects.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/química , Ácidos Nucleicos Heteroduplexes/química , Oligodesoxirribonucleotídeos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/farmacologia , Dicroísmo Circular , DNA Topoisomerases Tipo I/metabolismo , Desoxirribonucleases/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Estabilidade de Medicamentos , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Células MCF-7 , Espectroscopia de Ressonância Magnética , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ressonância de Plasmônio de Superfície , Timina/química , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia
13.
Biochem J ; 477(18): 3567-3582, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32886094

RESUMO

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.


Assuntos
Proteínas de Homeodomínio/química , Ácidos Nucleicos Heteroduplexes/química , Motivos de Aminoácidos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Domínios Proteicos , Relação Estrutura-Atividade , Recombinação V(D)J
14.
Methods Mol Biol ; 2176: 113-119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865786

RESUMO

Heteroduplex oligonucleotides (HDOs) were a novel type of nucleic acid drugs based on an antisense oligonucleotide (ASO) strand and its complementary RNA (cRNA ) strand. HDOs were originally designed to improve the properties of RNase H-dependent ASOs and we reported in our first paper that HDOs conjugated with an α-tocopherol ligand (Toc-HDO ) based on a gapmer ASO showed 20 times higher silencing effect to liver apolipoprotein B (apoB) mRNA in vivo than the parent ASO. Thereafter the HDO strategy was found to be also effective for improving the properties of ASOs modulating blood-brain barrier function and ASO antimiRs which are RNase H-independent ASOs. Therefore, the HDO strategy has been shown to be versatile technology platform to develop effective nucleic acid drugs.


Assuntos
Inativação Gênica/efeitos dos fármacos , Ácidos Nucleicos Heteroduplexes/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA/farmacologia , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Terapia Genética/métodos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/uso terapêutico , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/uso terapêutico , RNA/química , RNA/uso terapêutico
15.
Nucleic Acids Res ; 48(17): 9899-9917, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32821947

RESUMO

Expansion of the GAA/TTC repeats in the first intron of the FXN gene causes Friedreich's ataxia. Non-canonical structures are linked to this expansion. DNA triplexes and R-loops are believed to arrest transcription, which results in frataxin deficiency and eventual neurodegeneration. We present a systematic in silico characterization of the possible DNA triplexes that could be assembled with GAA and TTC strands; the two hybrid duplexes [r(GAA):d(TTC) and d(GAA):r(UUC)] in an R-loop; and three hybrid triplexes that could form during bidirectional transcription when the non-template DNA strand bonds with the hybrid duplex (collapsed R-loops, where the two DNA strands remain antiparallel). For both Y·R:Y and R·R:Y DNA triplexes, the parallel third strand orientation is more stable; both parallel and antiparallel protonated d(GA+A)·d(GAA):d(TTC) triplexes are stable. Apparent contradictions in the literature about the R·R:Y triplex stability is probably due to lack of molecular resolution, since shifting the third strand by a single nucleotide alters the stability ranking. In the collapsed R-loops, antiparallel d(TTC+)·d(GAA):r(UUC) is unstable, while parallel d(GAA)·r(GAA):d(TTC) and d(GA+A)·r(GAA):d(TTC) are stable. In addition to providing new structural perspectives for specific therapeutic aims, our results contribute to a systematic structural basis for the emerging field of quantitative R-loop biology.


Assuntos
DNA/química , Ataxia de Friedreich/genética , Ácidos Nucleicos Heteroduplexes/química , Repetições de Trinucleotídeos , Humanos , Estruturas R-Loop
16.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

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.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
17.
Mol Cell ; 78(6): 1252-1263.e3, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32362315

RESUMO

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.


Assuntos
Troca Genética/fisiologia , Recombinação Homóloga/fisiologia , Meiose/fisiologia , Animais , Segregação de Cromossomos/genética , Troca Genética/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Recombinação Homóloga/genética , Masculino , Meiose/genética , Camundongos , Camundongos Endogâmicos DBA , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ácidos Nucleicos Heteroduplexes/genética
18.
Nucleic Acids Res ; 48(12): 6954-6969, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32459314

RESUMO

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.


Assuntos
Enzimas de Restrição do DNA/ultraestrutura , DNA/ultraestrutura , Ácidos Nucleicos Heteroduplexes/ultraestrutura , RNA/ultraestrutura , Anabaena variabilis/genética , Sítios de Ligação/genética , Cristalografia por Raios X , DNA/genética , Quebras de DNA de Cadeia Dupla , Enzimas de Restrição do DNA/genética , Metais/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , RNA/genética
19.
Int J Mol Sci ; 21(5)2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32131532

RESUMO

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.


Assuntos
DNA/química , Instabilidade Genômica , Ácidos Nucleicos Heteroduplexes/química , Ribonucleotídeos/química , Animais , DNA/genética , Replicação do DNA , Humanos , Ácidos Nucleicos Heteroduplexes/genética , Estruturas R-Loop , Ribonucleotídeos/genética
20.
Nat Chem Biol ; 16(5): 489-492, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32015521

RESUMO

RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.


Assuntos
Aldeídos/química , RNA/química , Animais , Butanonas , Células-Tronco Embrionárias , Guanina/química , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes , Dobramento de RNA , Transcriptoma
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