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1.
Mol Biol Rep ; 51(1): 628, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38717629

RESUMO

Autoinduction systems in Escherichia coli can control the production of proteins without the addition of a particular inducer. In the present study, we optimized the heterologous expression of Moloney Murine Leukemia Virus derived Reverse Transcriptase (MMLV-RT) in E. coli. Among 4 autoinduction media, media Imperial College resulted the highest MMLV-RT overexpression in E. coli BL21 Star (DE3) with incubation time 96 h. The enzyme was produced most optimum in soluble fraction of lysate cells. The MMLV-RT was then purified using the Immobilized Metal Affinity Chromatography method and had specific activity of 629.4 U/mg. The system resulted lower specific activity and longer incubation of the enzyme than a classical Isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction system. However, the autoinduction resulted higher yield of the enzyme than the conventional induction (27.8%). Techno Economic Analysis revealed that this method could produce MMLV-RT using autoinduction at half the cost of MMLV-RT production by IPTG-induction. Bioprocessing techniques are necessary to conduct to obtain higher quality of MMLV-RT under autoinduction system.


Assuntos
Escherichia coli , Vírus da Leucemia Murina de Moloney , DNA Polimerase Dirigida por RNA , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , DNA Polimerase Dirigida por RNA/genética , Isopropiltiogalactosídeo/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Meios de Cultura
2.
Viruses ; 16(4)2024 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-38675856

RESUMO

CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.


Assuntos
Bacteriófagos , Genoma Viral , Filogenia , Bacteriófagos/genética , Bacteriófagos/classificação , Proteínas Virais/genética , Proteínas Virais/metabolismo , Retroelementos , Variação Genética , Prófagos/genética , DNA Viral/genética , DNA Primase/genética , DNA Primase/metabolismo , Genômica/métodos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
3.
Nucleic Acids Res ; 52(8): 4723-4738, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38587192

RESUMO

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.


Assuntos
Bacteriófagos , Lactococcus , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófagos/genética , Cristalografia por Raios X , Lactococcus/virologia , Lactococcus/genética , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , DNA Polimerase Dirigida por RNA/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , Relação Estrutura-Atividade
4.
Sheng Wu Gong Cheng Xue Bao ; 40(3): 812-820, 2024 Mar 25.
Artigo em Chinês | MEDLINE | ID: mdl-38545979

RESUMO

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.


Assuntos
Colicinas , Taq Polimerase/genética , Taq Polimerase/química , Taq Polimerase/metabolismo , Colicinas/genética , Colicinas/metabolismo , Escherichia coli/metabolismo , DNA , Exonucleases , DNA Polimerase Dirigida por RNA/metabolismo , Thermus/genética , Thermus/metabolismo
5.
Trends Cancer ; 10(4): 286-288, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38499453

RESUMO

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Transcrição Reversa , Humanos , Células HeLa , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo
6.
Genes (Basel) ; 15(2)2024 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-38397133

RESUMO

L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.


Assuntos
Fenômenos Biológicos , Elementos Nucleotídeos Longos e Dispersos , Humanos , Animais , Elementos Nucleotídeos Longos e Dispersos/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Células HeLa , Endonucleases/genética , Telômero/genética , Telômero/metabolismo , Reparo do DNA/genética , Mamíferos/genética
7.
Int J Mol Sci ; 25(3)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38339086

RESUMO

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.


Assuntos
Infecções por HIV , HIV-1 , Produtos do Gene pol do Vírus da Imunodeficiência Humana , Humanos , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Protease de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Poliproteínas/genética , DNA Polimerase Dirigida por RNA/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/química
8.
Recent Pat Biotechnol ; 18(1): 71-83, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37016518

RESUMO

INTRODUCTION: Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. AIM: As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. METHODS: RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. CONCLUSION: This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.


Assuntos
Vírus da Leucemia Murina de Moloney , DNA Polimerase Dirigida por RNA , Animais , Camundongos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Proteínas de Transporte , DNA Arqueal , Patentes como Assunto , Proteínas de Ligação a DNA/metabolismo
9.
Nature ; 626(7997): 186-193, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096901

RESUMO

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.


Assuntos
DNA Complementar , Elementos Nucleotídeos Longos e Dispersos , RNA , Retroelementos , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , DNA Complementar/biossíntese , DNA Complementar/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , RNA/química , RNA/genética , RNA/metabolismo , Domínio Catalítico , Endonucleases/química , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Terapia Genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , DNA Polimerase Dirigida por RNA/ultraestrutura , DNA de Cadeia Simples/metabolismo , Quebras de DNA
10.
Nature ; 626(7997): 194-206, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096902

RESUMO

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.


Assuntos
Endonucleases , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Cristalografia por Raios X , DNA/biossíntese , DNA/genética , Imunidade Inata , Interferons/biossíntese
11.
Indian J Pathol Microbiol ; 66(4): 714-719, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38084521

RESUMO

Classifying diffuse large B cell lymphomas, not otherwise specified (DLBCL, NOS), is based on their cell-of-origin (COO) which is included in the WHO classification (2016), is essential to characterize them better in context of prognostication. While gene expression profiling (GEP) considered the gold standard and more recently, the Nanostring-based approach, classify these tumors accurately, many laboratories with limited resources and instrumentation need an alternate approach that is reliable, inexpensive, and with a reasonable turnaround. The Reverse Transcriptase Multiplex Ligation Dependant Probe Amplification (RT-MLPA) to subtype DLBCL, NOS cases, as designed by CALYM group appears to provide a good alternative but needs to be validated in other centres. Therefore, this study evaluated DLBCL, NOS and compared the results of RT-MLPA to that obtained by immunohistochemistry using the Hans algorithm. Materials and Methods: Sixty-five DLBCL, NOS cases were included and the RT-MLPA was set up and standardized using probes that were designed by the CALYM study group. Briefly, RNA was extracted converted to cDNA and the 21-gene expression classifier that also included probes to detect MYD88 mutations and EBER mRNA was performed by MLPA. The results were analyzed by the open home grown software designed by the same group and compared to the results obtained by IHC. Results: Forty-four of the sixty-five cases provided concordant results (k = 0.35) and if the MYD88 results were to be used as a classifier the concordance would have improved from 67.7% to 82%. The 21 discordant cases were divided into five categories to provide a possible explanation for the discordance. Further 26% and 31% of the samples tested were positive for MYD88 mutations and EBER mRNA, respectively. The test had a turnaround of three days. Conclusion: The test provided moderate (67.7%) concordance when compared with IHC and perhaps would have provided higher concordance if compared with GEP. The test also has the advantage of providing information on the MYD88 and EBV infection status. It was found to be reliable, easy to perform and standardize, requiring only routine instruments available in most molecular laboratories. The RT-MLPA assay therefore provides an alternative for laboratories that would require subtyping of DLBCL, NOS cases in the absence of an access to GEP or other instrument intensive methods.


Assuntos
Linfoma Difuso de Grandes Células B , DNA Polimerase Dirigida por RNA , Humanos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Perfilação da Expressão Gênica , RNA Mensageiro , Proteínas Adaptadoras de Transdução de Sinal/genética , Prognóstico
12.
Methods Enzymol ; 691: 17-27, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37914445

RESUMO

Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, robust method for direct RNA sequence analysis. The guidance for troubleshooting and further optimization are also provided.


Assuntos
DNA Polimerase Dirigida por RNA , RNA , RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA
13.
Methods Enzymol ; 691: 3-15, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37914451

RESUMO

RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.


Assuntos
DNA Polimerase Dirigida por RNA , RNA , RNA/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , Reação em Cadeia da Polimerase , Íntrons
14.
Adv Exp Med Biol ; 1415: 109-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440022

RESUMO

Prime editing (PE) is a novel, double-strand break (DSB)-independent gene editing technology that represents an exciting avenue for the treatment of inherited retinal diseases (IRDs). Given the extensive and heterogenous nature of the 280 genes associated with IRDs, genome editing has presented countless complications. However, recent advances in genome editing technologies have identified PE to have tremendous potential, with the capability to ameliorate small deletions and insertions in addition to all twelve possible transition and transversion mutations. The current PE system is based on the fusion of the Streptococcus pyogenes Cas9 (SpCas9) nickase H840A mutant and an optimized Moloney murine leukemia virus (MMLV) reverse-transcriptase (RT) in conjunction with a PE guide RNA (pegRNA). In this study, we developed a prime editor based on the avian myeloblastosis virus (AMV)-RT and showed its applicability for the installation of the PRPH2 c.828+1G>A mutation in HEK293 cells.


Assuntos
Vírus da Mieloblastose Aviária , DNA Polimerase Dirigida por RNA , Humanos , Animais , Camundongos , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Mieloblastose Aviária/genética , Vírus da Mieloblastose Aviária/metabolismo , Células HEK293 , Edição de Genes , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/metabolismo , Sistemas CRISPR-Cas
15.
Nat Struct Mol Biol ; 30(7): 878-890, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37400652

RESUMO

Telomerase is a special reverse transcriptase ribonucleoprotein dedicated to the synthesis of telomere repeats that protect chromosome ends. Among reverse transcriptases, telomerase is unique in using a stably associated RNA with an embedded template to synthesize a specified sequence. Moreover, it is capable of iteratively copying the same template region (repeat addition processivity) through multiple rounds of RNA-DNA unpairing and reannealing, that is, the translocation reaction. Biochemical analyses of telomerase over the past 3 decades in protozoa, fungi and mammals have identified structural elements that underpin telomerase mechanisms and have led to models that account for the special attributes of telomerase. Notably, these findings and models can now be interpreted and adjudicated through recent cryo-EM structures of Tetrahymena and human telomerase holoenzyme complexes in association with substrates and regulatory proteins. Collectively, these structures reveal the intricate protein-nucleic acid interactions that potentiate telomerase's unique translocation reaction and clarify how this enzyme reconfigures the basic reverse transcriptase scaffold to craft a polymerase dedicated to the synthesis of telomere DNA. Among the many new insights is the resolution of the telomerase 'anchor site' proposed more than 3 decades ago. The structures also highlight the nearly universal conservation of a protein-protein interface between an oligonucleotide/oligosaccharide-binding (OB)-fold regulatory protein and the telomerase catalytic subunit, which enables spatial and temporal regulation of telomerase function in vivo. In this Review, we discuss key features of the structures in combination with relevant functional analyses. We also examine conserved and divergent aspects of telomerase mechanisms as gleaned from studies in different model organisms.


Assuntos
Ácidos Nucleicos , Telomerase , Animais , Humanos , Telomerase/química , Telômero/metabolismo , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA/metabolismo , DNA , Mamíferos/genética
16.
Nature ; 620(7972): 218-225, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37438532

RESUMO

Retrotransposons are highly enriched in the animal genome1-3. The activation of retrotransposons can rewrite host DNA information and fundamentally impact host biology1-3. Although developmental activation of retrotransposons can offer benefits for the host, such as against virus infection, uncontrolled activation promotes disease or potentially drives ageing1-5. After activation, retrotransposons use their mRNA as templates to synthesize double-stranded DNA for making new insertions in the host genome1-3,6. Although the reverse transcriptase that they encode can synthesize the first-strand DNA1-3,6, how the second-strand DNA is generated remains largely unclear. Here we report that retrotransposons hijack the alternative end-joining (alt-EJ) DNA repair process of the host for a circularization step to synthesize their second-strand DNA. We used Nanopore sequencing to examine the fates of replicated retrotransposon DNA, and found that 10% of them achieve new insertions, whereas 90% exist as extrachromosomal circular DNA (eccDNA). Using eccDNA production as a readout, further genetic screens identified factors from alt-EJ as essential for retrotransposon replication. alt-EJ drives the second-strand synthesis of the long terminal repeat retrotransposon DNA through a circularization process and is therefore necessary for eccDNA production and new insertions. Together, our study reveals that alt-EJ is essential in driving the propagation of parasitic genomic retroelements. Our study uncovers a conserved function of this understudied DNA repair process, and provides a new perspective to understand-and potentially control-the retrotransposon life cycle.


Assuntos
Reparo do DNA por Junção de Extremidades , Replicação do DNA , DNA Circular , Parasitos , Retroelementos , Animais , Retroelementos/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Moldes Genéticos , DNA Circular/biossíntese , DNA Circular/genética , DNA Circular/metabolismo , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Parasitos/genética , Genoma/genética
17.
Nucleic Acids Res ; 51(14): 7125-7142, 2023 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-37279911

RESUMO

The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.


Reverse transcriptases (RTs) are replicative enzymes that copy RNA into DNA and undertake roles, including viral replication, retrotransposition and telomere maintenance. The initiation of RT synthesis activities is usually dependent on the presence of a primer. The current dogma proposes that a variety of indirect, RT-independent, priming mechanisms instigate synthesis. However, this study establishes that CRISPR-associated RTs (CARTs) are capable of priming DNA synthesis from scratch, which enables the capture of foreign genetic material for storage in CRISPR arrays. The authors also report that other notable RT family members, including retrotransposon RTs, telomerase and retroviral RT are, surprisingly, able to directly catalyze primer synthesis. These findings significantly alter our understanding of priming mechanisms utilised by RTs in various biological pathways.


Assuntos
DNA Polimerase Dirigida por RNA , DNA Polimerase Dirigida por DNA/genética , Transcriptase Reversa do HIV/genética , Íntrons/genética , Retroviridae/genética , RNA/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Replicação do DNA
18.
Cell ; 186(13): 2865-2879.e20, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37301196

RESUMO

Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.


Assuntos
RNA , Retroelementos , RNA/metabolismo , Clivagem do DNA , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa
19.
Retrovirology ; 20(1): 5, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37127613

RESUMO

BACKGROUND: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro. RESULTS: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels. CONCLUSIONS: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells.


Assuntos
HIV-1 , Proteínas Monoméricas de Ligação ao GTP , Humanos , HIV-1/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Fosforilação , Células U937 , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo
20.
RNA ; 29(8): 1301-1315, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37192815

RESUMO

Systematic evolution of ligands through exponential enrichment (SELEX) is widely used to identify functional nucleic acids, such as aptamers and ribozymes. Ideally, selective pressure drives the enrichment of sequences that display the function of interest (binding, catalysis, etc.). However, amplification biases from reverse transcription can overwhelm this enrichment and leave some functional sequences at a disadvantage, with cumulative effects across multiple rounds of selection. Libraries that are designed to include structural scaffolds can improve selection outcomes by sampling sequence space more strategically, but they are also susceptible to such amplification biases, particularly during reverse transcription. Therefore, we tested five reverse transcriptases (RTs)-ImProm-II, Marathon RT (MaRT), TGIRT-III, SuperScript IV (SSIV), and BST 3.0 DNA polymerase (BST)-to determine which enzymes introduced the least bias. We directly compared cDNA yield and processivity for these enzymes on RNA templates with varying degrees of structure under various reaction conditions. In these analyses, BST exhibited excellent processivity, generated large quantities of the full-length cDNA product, displayed little bias among templates with varying structure and sequence, and performed well on long, highly structured viral RNAs. Additionally, six RNA libraries containing either strong, moderate, or no incorporated structural elements were pooled and competed head-to-head in six rounds of an amplification-only selection without external selective pressure using either SSIV, ImProm-II, or BST during reverse transcription. High-throughput sequencing established that BST maintained the most neutral enrichment values, indicating low interlibrary bias over the course of six rounds, relative to SSIV and ImProm-II, and it introduced minimal mutational bias.


Assuntos
Aptâmeros de Nucleotídeos , Transcrição Reversa , DNA Complementar , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Biblioteca Gênica , RNA Viral , Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros
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