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1.
PLoS One ; 15(2): e0228774, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32069311

RESUMO

Ribonuclease H2 (RNase H2) exhibits both single ribonucleotide excision activity (activity A) and RNA strand degrading activity (activity B). Val143 of human RNase H2 is located at the active site and is conserved in eukaryotic RNase H2. In this study, we explored the role of Val143 in catalytic activity and substrate specificity. Nineteen single variants at amino acid position 143 were expressed in E. coli, and all variants except for V143C and V143M were purified from the cells. When the activity of the wild-type human RNase H2 (WT) was set as 100%, the relative activities A and B of the 17 variants were in the range of 0.05-130 and 0.02-42%, respectively. When the ratio of the relative activity A to the relative activity B of WT was set as 1, the ratios of the 17 variants were in the range of 0.2-5.7. This indicates that valine is optimal for balancing the two activities. The ratios for V143Y and V143W were relatively high (5.6 and 5.5, respectively), suggesting that the bulky residues like tyrosine and tryptophan at position 143 caused steric hindrance with the 2'-OH of the sugar moiety of the ribonucleotide at the 5' side of the scissile phosphodiester bond. The ratio for V143Q was relatively low (0.2). These results suggested that Val143 is not critical for, but plays a role in determining catalytic activity and substrate specificity.


Assuntos
Biocatálise , Ribonuclease H/química , Ribonuclease H/metabolismo , Valina , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Modelos Moleculares , Mutação , Ribonuclease H/genética , Especificidade por Substrato
2.
Mol Cell ; 77(5): 1032-1043.e4, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31924447

RESUMO

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.


Assuntos
Cromatina/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Terminação da Transcrição Genética , Animais , Cromatina/genética , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Células HCT116 , Humanos , Íntrons , Camundongos Endogâmicos C57BL , Modelos Genéticos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonuclease H/genética , Fatores de Tempo
3.
Chemistry ; 26(6): 1368-1379, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31682037

RESUMO

Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNase H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNase H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.


Assuntos
Inativação Gênica , Nucleotídeos/química , Oligonucleotídeos Antissenso/química , Ribonuclease H/metabolismo , Sequência de Bases , Estabilidade Enzimática , Células HeLa , Humanos , Concentração Inibidora 50 , Desnaturação de Ácido Nucleico , Nucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA/química , RNA/metabolismo , Temperatura , Transfecção
4.
Genes Dev ; 34(1-2): 132-145, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31805520

RESUMO

The allosteric and torpedo models have been used for 30 yr to explain how transcription terminates on protein-coding genes. The former invokes termination via conformational changes in the transcription complex and the latter proposes that degradation of the downstream product of poly(A) signal (PAS) processing is important. Here, we describe a single mechanism incorporating features of both models. We show that termination is completely abolished by rapid elimination of CPSF73, which causes very extensive transcriptional readthrough genome-wide. This is because CPSF73 functions upstream of modifications to the elongation complex and provides an entry site for the XRN2 torpedo. Rapid depletion of XRN2 enriches these events that we show are underpinned by protein phosphatase 1 (PP1) activity, the inhibition of which extends readthrough in the absence of XRN2. Our results suggest a combined allosteric/torpedo mechanism, in which PP1-dependent slowing down of polymerases over termination regions facilitates their pursuit/capture by XRN2 following PAS processing.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Terminação da Transcrição Genética/fisiologia , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/genética , Exorribonucleases/metabolismo , Deleção de Genes , Células HCT116 , Humanos , RNA/metabolismo , RNA Polimerase II/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Ribonuclease H/metabolismo
5.
Nucleic Acids Res ; 48(1): 63-74, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31754711

RESUMO

The introduction of non-bridging phosphorothioate (PS) linkages in oligonucleotides has been instrumental for the development of RNA therapeutics and antisense oligonucleotides. This modification offers significantly increased metabolic stability as well as improved pharmacokinetic properties. However, due to the chiral nature of the phosphorothioate, every PS group doubles the amount of possible stereoisomers. Thus PS oligonucleotides are generally obtained as an inseparable mixture of a multitude of diastereoisomeric compounds. Herein, we describe the introduction of non-chiral 3' thiophosphate linkages into antisense oligonucleotides and report their in vitro as well as in vivo activity. The obtained results are carefully investigated for the individual parameters contributing to antisense activity of 3' and 5' thiophosphate modified oligonucleotides (target binding, RNase H recruitment, nuclease stability). We conclude that nuclease stability is the major challenge for this approach. These results highlight the importance of selecting meaningful in vitro experiments particularly when examining hitherto unexplored chemical modifications.


Assuntos
Apolipoproteína B-100/genética , Oligonucleotídeos/genética , Fosfatos/química , Oligonucleotídeos Fosforotioatos/genética , RNA Longo não Codificante/genética , Animais , Apolipoproteína B-100/antagonistas & inibidores , Apolipoproteína B-100/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Fosfatos/metabolismo , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Estereoisomerismo
6.
Chem Commun (Camb) ; 56(4): 639-642, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31833487

RESUMO

In this work, we have developed a novel DNAzyme motor initiated by endogenous enzyme for sensitive imaging of intracellular RNase H activity.


Assuntos
DNA Catalítico/metabolismo , Imagem Óptica , Ribonuclease H/metabolismo , Sobrevivência Celular , Células HeLa , Células Hep G2 , Humanos , Células MCF-7 , Microscopia Confocal , Microscopia de Fluorescência
7.
Mol Cell ; 76(4): 600-616.e6, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31679819

RESUMO

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.


Assuntos
Genoma Humano , Regiões Promotoras Genéticas , Estruturas R-Loop , RNA Antissenso/biossíntese , RNA Longo não Codificante/biossíntese , Transcrição Genética , Ativação Transcricional , Células HEK293 , Células HeLa , Humanos , RNA Antissenso/genética , RNA Longo não Codificante/genética , Ribonuclease H/metabolismo , Relação Estrutura-Atividade
9.
DNA Repair (Amst) ; 84: 102736, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31761672

RESUMO

Eukaryotic RNases H2 have dual functions in initiating the removal of ribonucleoside monophosphates (rNMPs) incorporated by DNA polymerases during DNA synthesis and in cleaving the RNA moiety of RNA/DNA hybrids formed during transcription and retrotransposition. The other major cellular RNase H, RNase H1, shares the hybrid processing activity, but not all substrates. After RNase H2 incision at the rNMPs in DNA the Ribonucleotide Excision Repair (RER) pathway completes the removal, restoring dsDNA. The development of the RNase H2-RED (Ribonucleotide Excision Defective) mutant enzyme, which can process RNA/DNA hybrids but is unable to cleave rNMPs embedded in DNA has unlinked the two activities and illuminated the roles of RNase H2 in cellular metabolism. Studies mostly in Saccharomyces cerevisiae, have shown both activities of RNase H2 are necessary to maintain genome integrity and that RNase H1 and H2 have overlapping as well as distinct RNA/DNA hybrid substrates. In mouse RNase H2-RED confirmed that rNMPs in DNA during embryogenesis induce lethality in a p53-dependent DNA damage response. In mammalian cell cultures, RNase H2-RED helped identifying DNA lesions produced by Top1 cleavage at rNMPs and led to determine that RNase H2 participates in the retrotransposition of LINE-1 elements. In this review, we summarize the studies and conclusions reached by utilization of RNase H2-RED enzyme in different model systems.


Assuntos
Reparo do DNA , Ribonuclease H/metabolismo , Animais , Humanos , Ribonuclease H/química , Ribonuclease H/genética , Ribonucleotídeos/genética
10.
J Biochem ; 166(6): 537-545, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31529068

RESUMO

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.


Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Malformações do Sistema Nervoso/genética , Ribonuclease H/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Humanos , Mutação , Malformações do Sistema Nervoso/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo
11.
Nucleic Acids Res ; 47(20): 10865-10880, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31495875

RESUMO

The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease H/metabolismo , Domínio Catalítico , Nucléolo Celular/metabolismo , Sobrevivência Celular , Proteínas de Ligação a DNA/química , Células HEK293 , Células HeLa , Humanos , Cinética , Ligação Proteica , Proteínas de Ligação a RNA/química , Ribonuclease H/química
12.
DNA Repair (Amst) ; 84: 102693, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31471263

RESUMO

DNA replication, the faithful copying of genetic material, must be tightly regulated to produce daughter cells with intact copies of the chromosome(s). This regulated replication is initiated by binding of specific proteins at replication origins, such as DnaA to oriC in bacteria. However, unregulated replication can sometimes be initiated at other sites, which can threaten genomic stability. One of the first systems of unregulated replication to be described is the one activated in Escherichia coli mutants lacking RNase HI (rnhA). In fact, rnhA mutants can replicate their chromosomes in a DnaA- and oriC-independent process. Because this replication occurs in cells lacking RNase HI, it is proposed that RNA from R-loops is used as a DNA polymerase primer. Replication from R-loops has recently attracted increased attention due to the advent of DNA:RNA hybrid immunoprecipitation coupled with high-throughput DNA sequencing that revealed the high prevalence of R-loop formation in many organisms, and the demonstration that R-loops can severely threaten genomic stability. Although R-loops have been linked to genomic instability mostly via replication stress, evidence of their toxic effects via unregulated replication has also been presented. Replication from R-loops may also beneficially trigger stress-induced mutagenesis (SIM) that assists bacterial adaptation to stress. Here, we describe the cis- and trans-acting elements involved in R-loop-dependent replication in bacteria, with an emphasis on new data obtained with type 1A topoisomerase mutants and new available technologies. Furthermore, we discuss about the mechanism(s) by which R-loops can reshape the genome with both negative and positive outcomes.


Assuntos
Replicação do DNA , Genoma Bacteriano , Instabilidade Genômica , Estruturas R-Loop , Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Ribonuclease H/metabolismo
13.
Nanotechnology ; 30(46): 465502, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31426052

RESUMO

Huntington's disease is a chronic progressive neurodegeneration which is caused by CAG repeat sequences expanding in the huntingtin gene. There is currently no disease-modifying treatment for the disease, and its progression can only be slowed down before the onset of symptoms. A novel fluorescent platform which contains an RNA probe and graphene oxide for detection of the biomarker of Huntington's disease, CAG repeat sequences, was constructed in this investigation. In addition, RNase H was employed in the fluorescent system to enhance the sensitivity of the detection capability. The fluorescent signal was increased through the cyclic amplified reaction, which results from RNase H, specifically digestion of the RNA strand in the complement of the RNA-DNA duplex. The designed measurement method can detect CAG repeat sequences with a detection limit of 108 pM (R2 = 0.968) under which we optimized assay conditions. Furthermore, the detection limit is approximately 18 times lower than the traditional DNA and graphene oxide detection method without assistance of RNase H. Additionally, the probing platform also shows stronger ability to discriminate between the fluorescence of the target sequence and that of other non-target sequences. The results of our studies demonstrate that the RNase H amplified RNA probe and graphene oxide system exhibited excellent sensitivity and selectivity to the target of CAG repeats sequences.


Assuntos
Grafite/química , Doença de Huntington/diagnóstico , Sondas RNA/química , Ribonuclease H/metabolismo , Repetições de Trinucleotídeos , Diagnóstico Precoce , Marcadores Genéticos/genética , Humanos , Doença de Huntington/genética , Sensibilidade e Especificidade , Espectrometria de Fluorescência
14.
DNA Repair (Amst) ; 84: 102672, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31371183

RESUMO

RNases H are a family of endonucleases that hydrolyze RNA residues in various nucleic acids. These enzymes are present in all branches of life, and their counterpart domains are also found in reverse transcriptases (RTs) from retroviruses and retroelements. RNases H are divided into two main classes (RNases H1 and H2 or type 1 and type 2 enzymes) with common structural features of the catalytic domain but different range of substrates for enzymatic cleavage. Additionally, a third class is found in some Archaea and bacteria. Besides distinct cellular functions specific for each type of RNases H, this family of proteins is generally involved in the maintenance of genome stability with overlapping and cooperative role in removal of R-loops thus preventing their accumulation. Extensive biochemical and structural studies of RNases H provided not only a comprehensive and complete picture of their mechanism but also revealed key basic principles of nucleic acid recognition and processing. RNase H1 is present in prokaryotes and eukaryotes and cleaves RNA in RNA/DNA hybrids. Its main function is hybrid removal, notably in the context of R-loops. RNase H2, which is also present in all branches of life, can play a similar role but it also has a specialized function in the cleavage of single ribonucleotides embedded in the DNA. RNase H3 is present in Archaea and bacteria and is closely related to RNase H2 in sequence and structure but has RNase H1-like biochemical properties. This review summarizes the mechanisms of substrate recognition and enzymatic cleavage by different classes of RNases H with particular insights into structural features of nucleic acid binding, specificity towards RNA and/or DNA strands and catalysis.


Assuntos
Ribonuclease H/química , Animais , Bactérias/enzimologia , Bactérias/genética , Domínio Catalítico , Sequência Conservada , Humanos , Estruturas R-Loop , Ribonuclease H/genética , Ribonuclease H/metabolismo , Especificidade por Substrato
15.
Mol Cell ; 76(1): 44-56.e3, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31444105

RESUMO

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.


Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Evolução Molecular , Inosina/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Animais , Proteínas Argonauta/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Humanos , Magnésio/metabolismo , Manganês/metabolismo , Camundongos , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease H/genética , Relação Estrutura-Atividade , Especificidade por Substrato
16.
Nat Protoc ; 14(8): 2571-2594, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31341290

RESUMO

RNase H-dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2-4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d.


Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Células Cultivadas , Clonagem Molecular , Humanos , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease H/metabolismo , Linfócitos T/química , Linfócitos T/citologia
17.
J Biol Chem ; 294(35): 13061-13072, 2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31300556

RESUMO

The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.


Assuntos
DNA/metabolismo , Escherichia coli/metabolismo , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismo , Ribosemonofosfatos/metabolismo , Células HeLa , Humanos , Oxirredução , Células Tumorais Cultivadas
18.
DNA Repair (Amst) ; 84: 102630, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31178343

RESUMO

The activity and specificity of ribonuclease H1, RNase H1, has been known for over half a century; like all enzymes in its class, it degrades RNA only when it is hybridized to DNA. However, the essential role of RNase H1 in mitochondrial DNA maintenance was not recognized until 2003, and empirical evidence that it is required to process RNA primers of mitochondrial DNA had to wait until 2015. In the same year, mutations in the RNASEH1 gene were linked to human mitochondrial diseases. The most recent studies suggest that in addition to primer-processing, RNase H1 determines the fate of R-loops, although not primarily those that might present an obstacle to DNA replication, but ones that contribute to the organization of mitochondrial DNA and the unusual mechanism of replication in mitochondria that utilizes transcripts for the strand-asynchronous mechanism of mitochondrial DNA replication. A full understanding of the role of RNase H1 in mtDNA metabolism will depend on further study, including careful consideration of its ability to stabilize, as well as to degrade RNA/DNA hybrids, and its regulation by oxidation or other mechanisms. Nevertheless, RNase H1 is already staking a strong claim to be the most versatile factor involved in propagating the DNA in the mitochondria.


Assuntos
DNA Mitocondrial/genética , Ribonuclease H/metabolismo , Animais , DNA Mitocondrial/química , DNA Mitocondrial/metabolismo , Humanos , Doenças Mitocondriais/genética , Estruturas R-Loop , Ribonuclease H/genética
19.
DNA Repair (Amst) ; 84: 102633, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31231063

RESUMO

An R-loop is a structure that forms when an RNA transcript stays bound to the DNA strand that encodes it and leaves the complementary strand exposed as a loop of single stranded DNA. R-loops accumulate when the processing of RNA transcripts is impaired. The failure to remove these RNA-DNA hybrids can lead to replication fork stalling and genome instability. Resolution of R-loops is thought to be mediated mainly by RNase H enzymes through the removal and degradation of the RNA in the hybrid. However, DNA helicases can also dismantle R-loops by displacing the bound RNA. In particular, the Pif1 family DNA helicases have been shown to regulate R-loop formation at specific genomic loci, such as tRNA genes and centromeres. Here we review the roles of Pif1 family helicases in vivo and in vitro and discuss evidence that Pif1 family helicases act on RNA-DNA hybrids and highlight their potential roles in complementing RNase H for R-loop resolution.


Assuntos
Estruturas R-Loop , Ribonuclease H/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Nat Commun ; 10(1): 2253, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138795

RESUMO

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Homeostase do Telômero/genética , Telômero/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , RecQ Helicases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
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