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1.
Mol Immunol ; 116: 63-72, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622795

RESUMO

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.


Assuntos
Citidina Desaminase/genética , DNA Topoisomerases Tipo I/genética , Genes de Imunoglobulinas/genética , Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Hipermutação Somática de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linhagem Celular , Núcleo Celular/genética , Galinhas/genética , Switching de Imunoglobulina , Imunoprecipitação/métodos , Camundongos , Isoformas de Proteínas/genética
2.
Mol Cell ; 75(6): 1286-1298.e12, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31473101

RESUMO

Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína de Replicação A/metabolismo , Animais , Células CHO , Cricetulus , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Células HEK293 , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Proteína de Replicação A/genética
3.
Nat Commun ; 10(1): 3612, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399578

RESUMO

Base editing tools for cytosine to thymine (C-T) conversion enable genome manipulation at single base-pair resolution with high efficiency. Available base editors (BEs) for C-T conversion (CBEs) have restricted editing scopes and nonnegligible off-target effects, which limit their applications. Here, by screening diversified lamprey cytidine deaminases, we establish various CBEs with expanded and diversified editing scopes, which could be further refined by various fusing strategies, fusing at either N-terminus or C-terminus of nCas9. Furthermore, off-target analysis reveals that several CBEs display improved fidelity. Our study expands the toolkits for C-T conversion, serves as guidance for appropriate choice and offers a framework for benchmarking future improvement of base editing tools.


Assuntos
Citidina Desaminase/genética , Citosina , Edição de Genes/métodos , Timina , Pareamento de Bases , Sequência de Bases , Sistemas CRISPR-Cas , Citidina Desaminase/classificação , Citidina Desaminase/metabolismo , Células HCT116 , Células HEK293 , Humanos
4.
Nucleic Acids Res ; 47(18): 9666-9684, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31392335

RESUMO

Break induced replication (BIR) is a double strand break repair pathway that can promote genetic instabilities similar to those observed in cancer. Instead of a replication fork, BIR is driven by a migration bubble where asynchronous synthesis between leading and lagging strands leads to accumulation of single-stranded DNA (ssDNA) that promotes mutation. However, the details of the mechanism of mutagenesis, including the identity of the participating proteins, remain unknown. Using yeast as a model, we demonstrate that mutagenic ssDNA is formed at multiple positions along the BIR track and that Pol ζ is responsible for the majority of both spontaneous and damage-induced base substitutions during BIR. We also report that BIR creates a potent substrate for APOBEC3A (A3A) cytidine deaminase that can promote formation of mutation clusters along the entire track of BIR. Finally, we demonstrate that uracil glycosylase initiates the bypass of DNA damage induced by A3A in the context of BIR without formation of base substitutions, but instead this pathway frequently leads to chromosomal rearrangements. Together, the expression of A3A during BIR in yeast recapitulates the main features of APOBEC-induced kataegis in human cancers, suggesting that BIR might represent an important source of these hyper-mutagenic events.


Assuntos
Cromossomos/genética , Citidina Desaminase/genética , Reparo do DNA/genética , Proteínas/genética , Recombinação Genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Humanos , Mutagênese/genética , Mutação , Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
5.
Mol Med Rep ; 20(3): 2177-2188, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322199

RESUMO

The present study examined the relationships between the single nucleotide polymorphisms (SNPs) of three members of the apolipoprotein B mRNA­editing catalytic polypeptide­like 3 (A3) gene family, A3A, A3B and A3H, and hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) in a Han Chinese population. A total of 654 patients were enrolled in the study between January 2012 and July 2016, including 104 patients with chronic HBV infection (CHB), 265 patients with HBV­related liver cirrhosis and 285 patients with HBV­related HCC. A total of two A3A SNPs (rs7286317 and rs7290153), three A3B SNPs (rs2267398, rs2267401 and rs2076109), and five A3H SNPs (rs56695217, rs139302, rs139297, rs139316 and rs139292) were genotyped using a MassArray system. Statistical analysis and haplotype estimation were conducted using Haploview and Unphased software. No significant associations were observed between the A3A, A3B and A3H SNPs and the development of CHB and HCC. Haplotype analysis revealed that the mutant haplotypes C­T­A, C­T­G, T­G­G and T­T­G from the A3B SNPs rs2267398­rs2267401­rs2076109 carried a lower risk of HCC than the reference haplotype. These findings suggested that there was no relationship between A3A, A3B and A3H SNPs and CHB progression or HCC development in the Han Chinese population.


Assuntos
Aminoidrolases/genética , Carcinoma Hepatocelular/genética , Citidina Desaminase/genética , Hepatite B Crônica/genética , Neoplasias Hepáticas/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/virologia , China/epidemiologia , Feminino , Predisposição Genética para Doença , Hepatite B Crônica/complicações , Hepatite B Crônica/epidemiologia , Humanos , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único
6.
Mol Immunol ; 112: 198-205, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31176199

RESUMO

AID initiates both somatic hypermutation (SHM) and class switch recombination (CSR) in Ig genes. AID-induced mutations are linked with transcription initiation and elongation. Transcription occurs in the context of chromatin and thus RNA PolII and AID need to deal with nucleosomes. Both nucleosome stability and positioning significantly influence the accessibility of AID to Ig genes and the SHM pattern. Interestingly, in the nucleosome, SHM process seems to have a preference for the top strand. To know whether the preferential targeting of SHM to the top strand is due to a post-AID event, we expressed an inhibitor of Uracil DNA glycosylase (UNG), Ugi, into DT40 cells containing the nucleosome positioning sequence (MP2) and compared the SHM pattern. We observed a similar preference to the top strand for the high-affinity nucleosome positioning sequence in UNG inhibited cells. Furthermore, to understand whether the primary sequence of nucleosome sequence is influencing preferential targeting, we introduced two copies of MP2 sequence in the reverse orientation (MP2R) into a variable Ig gene. We observed that the MP2R cells also demonstrated preferential targeting of the non-transcribed strand in nucleosome as compared to the transcribed strand, confirming that in nucleosome sequences AID has better access to Cs on the top strand. The preferential targeting of AID on the top strand suggests that RNA Pol-II stalls while it transcribes the stable nucleosomes, thus giving ample opportunity for the transcribed strand to form R-loops with the nascent RNA, thereby gives limited access to AID on the bottom strand.


Assuntos
Citidina Desaminase/genética , Nucleossomos/genética , Animais , Linfócitos B/fisiologia , Linhagem Celular , Galinhas , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , RNA Polimerase II/genética , Hipermutação Somática de Imunoglobulina/genética , Uracila-DNA Glicosidase/genética
7.
Gut ; 68(10): 1846-1857, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31154396

RESUMO

OBJECTIVE: APOBEC3B (A3B), a cytidine deaminase acting as a contributor to the APOBEC mutation pattern in many kinds of tumours, is upregulated in patients with hepatocellular carcinoma (HCC). However, APOBEC mutation patterns are absent in HCC. The mechanism of how A3B affects HCC progression remains elusive. DESIGN: A3B -promoter luciferase reporter and other techniques were applied to elucidate mechanisms of A3B upregulation in HCC. A3B overexpression and knockdown cell models, immunocompetent and immune-deficient mouse HCC model were conducted to investigate the influence of A3B on HCC progression. RNA-seq, flow cytometry and other techniques were conducted to analyse how A3B modulated the cytokine to enhance the recruitment of myeloid--derived suppressor cells (MDSCs) and tumour--associated macrophages (TAMs). RESULTS: A3B upregulation through non-classical nuclear factor-κB (NF-κB)signalling promotes HCC growth in immunocompetent mice, associated with an increase of MDSCs, TAMs and programmed cell death1 (PD1) exprssed CD8+ T cells. A CCR2 antagonist suppressed TAMs and MDSCs infiltration and delayed tumour growth in A3B and A3BE68Q/E255Q- expressing mouse tumours. Mechanistically, A3B upregulation in HCC depresses global H3K27me3 abundance via interaction with polycomb repressor complex 2 (PRC2) and reduces an occupancy of H3K27me3 on promoters of the chemokine CCL2 to recruit massive TAMs and MDSCs. CONCLUSION: Our observations uncover a deaminase-independent role of the A3B in modulating the HCC microenvironment and demonstrate a proof for the concept of targeting A3B in HCC immunotherapy.


Assuntos
Carcinoma Hepatocelular/genética , Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas Experimentais/genética , Antígenos de Histocompatibilidade Menor/genética , Microambiente Tumoral/fisiologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Citidina Desaminase/biossíntese , DNA de Neoplasias/genética , Progressão da Doença , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/biossíntese , Regiões Promotoras Genéticas
8.
Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050

RESUMO

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
9.
PLoS Genet ; 15(6): e1007721, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31199803

RESUMO

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.


Assuntos
Linfócitos B/imunologia , Citidina Desaminase/genética , Região de Troca de Imunoglobulinas/genética , Imunoglobulinas/imunologia , Alelos , Animais , Diferenciação Celular/genética , Citidina Desaminase/imunologia , Marcação de Genes , Humanos , Região de Troca de Imunoglobulinas/imunologia , Tecido Linfoide/imunologia , Camundongos , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Sequências Reguladoras de Ácido Nucleico
10.
Nucleic Acids Res ; 47(14): 7418-7429, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127309

RESUMO

Affinity maturation of the humoral immune response depends on somatic hypermutation (SHM) of immunoglobulin (Ig) genes, which is initiated by targeted lesion introduction by activation-induced deaminase (AID), followed by error-prone DNA repair. Stringent regulation of this process is essential to prevent genetic instability, but no negative feedback control has been identified to date. Here we show that poly(ADP-ribose) polymerase-1 (PARP-1) is a key factor restricting AID activity during somatic hypermutation. Poly(ADP-ribose) (PAR) chains formed at DNA breaks trigger AID-PAR association, thus preventing excessive DNA damage induction at sites of AID action. Accordingly, AID activity and somatic hypermutation at the Ig variable region is decreased by PARP-1 activity. In addition, PARP-1 regulates DNA lesion processing by affecting strand biased A:T mutagenesis. Our study establishes a novel function of the ancestral genome maintenance factor PARP-1 as a critical local feedback regulator of both AID activity and DNA repair during Ig gene diversification.


Assuntos
Citidina Desaminase/genética , Genes de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Poli(ADP-Ribose) Polimerase-1/genética , Hipermutação Somática de Imunoglobulina/genética , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Células Cultivadas , Citidina Desaminase/metabolismo , Dano ao DNA , Reparo do DNA , Humanos , Camundongos , Mutação , Poli(ADP-Ribose) Polimerase-1/metabolismo
11.
Methods Cell Biol ; 151: 305-321, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948015

RESUMO

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs (gRNAs) of the CRISPR/Cas9 system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/Cas9 system efficiently and specifically targets sea urchin genes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9 protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminase to Cas9 (Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/Cas9 application in sea urchin embryos, including gRNA design, in vitro synthesis of single guide RNA (sgRNA), and the usages of the CRISPR/Cas9 technology for gene knockout and single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Ouriços-do-Mar/genética , Animais , Citidina Desaminase/genética , Marcação de Genes/métodos , Vetores Genéticos , Genoma/genética , Ouriços-do-Mar/crescimento & desenvolvimento
12.
Mol Biol Rep ; 46(3): 3333-3347, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980272

RESUMO

The aim of the present study is to determine the expression levels of PYHIN (IFI16 and AIM2) and APOBEC3 (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) gene family members in a cohort of patients with head and neck squamous cell carcinoma (HNSCC) and assess their potential correlation with human papillomavirus (HPV) infection status, clinical characteristics, and survival. For this purpose, 34 HNSCC tissue specimens along with healthy surrounding mucosa were collected from patients surgically treated for HNSCC. Nucleic acids were isolated to assess the presence of HPV and the expression levels of selected molecular markers. Survival analysis was carried out using the Kaplan-Meier method. In HPV-negative (HPV-) HNSCCs, we detected low mRNA expression levels of IFI16, A3A, and A3B, whereas these genes were upregulated of 2-100 folds in HPV-positive (HPV+) tumors (p < 0.05). Interestingly, AIM2 gene expression levels were predominantly unchanged in HPV+ HNSCCs compared to their HPV- counterparts, in which AIM2 was predominantly upregulated (10% vs. 50% of patients). In HPV- tumors, upregulation of TP53, NOTCH1, PD-L1, and IFI16 correlated with lower occurrence of nodal metastases. On the other hand, the expression of APOBEC family members did not correlate with clinical characteristics. Regarding survival, patients with upregulated A3F gene expression had a worse prognosis, while patients without changes in A3H expression had a lower survival rate. In conclusion, our findings indicate that the innate immune sensors IFI16 and AIM2 and some APOBEC family members could be potentially used as biomarkers for disease outcome in HNSCC patients regardless of HPV presence.


Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Proteínas Nucleares/genética , Papillomaviridae/isolamento & purificação , Fosfoproteínas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Adulto , Idoso , Biomarcadores Tumorais/genética , Estudos de Coortes , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Nucleares/metabolismo , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Fosfoproteínas/metabolismo , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida
13.
Nat Commun ; 10(1): 1234, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874552

RESUMO

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods provide new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we develop a genome editing strategy using a cytidine deaminase fused with nickase Cas9 (nCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns serve as a genetic barcode, which is induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.


Assuntos
Linhagem da Célula/genética , Código de Barras de DNA Taxonômico/métodos , Edição de Genes/métodos , Elementos Nucleotídeos Longos e Dispersos/genética , Proteína 9 Associada à CRISPR/genética , Diferenciação Celular/genética , Citidina Desaminase/genética , Células HEK293 , Células HeLa , Humanos , Mutagênese , RNA Guia/genética , Análise de Célula Única/métodos , Imagem com Lapso de Tempo
14.
J Phys Chem A ; 123(13): 3030-3037, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30848911

RESUMO

Activation-induced deoxycytidine deaminase (AID) is a key enzyme in the human immune system. AID binds to and catalyzes random point mutations on the immunoglobulin (Ig) gene, leading to diversification of the Ig gene sequence by random walk motions, scanning for cytidines and turning them to uracils. The mutation patterns deposited by AID on its substrate DNA sequences can be interpreted as random binary words, and the information content of this stochastically generated library of mutated DNA sequences can be measured by its entropy. In this paper, we derive an analytical formula for this entropy and show that the stochastic scanning + catalytic dynamics of AID is controlled by a characteristic length that depends on the diffusion coefficient of AID and the catalytic rate. Experiments showed that the deamination rates have a sequence context dependence, where mutations are generated at higher intensities on DNA sequences with higher densities of mutable sites. We derive an isomorphism between this classical system and a quantum mechanical model and use this isomorphism to explain why AID appears to focus its scanning on regions with higher concentrations of deaminable sites. Using path integral Monte Carlo simulations of the quantum isomorphic system, we demonstrate how AID's scanning indeed depends on the context of the DNA sequence and how this affects the entropy of the library of generated mutant clones. Examining detailed features in the entropy of the experimentally generated clone library, we provide clear evidence that the random walk of AID on its substrate DNA is focused near hot spots. The model calculations applied to the experimental data show that the observed per-site mutation frequencies display similar contextual dependences as observed in the experiments, in which hot motifs are located adjacent to several different types of hot and cold motifs.


Assuntos
Citidina Desaminase/metabolismo , Entropia , Teoria Quântica , Sequência de Aminoácidos , Biocatálise , Citidina Desaminase/química , Citidina Desaminase/genética , Difusão , Mutação , Processos Estocásticos
15.
N Biotechnol ; 51: 67-79, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30822538

RESUMO

AID/APOBEC3 enzymes are cytidine deaminases that mutate antibody and retroviral genes and also mediate extensive tumor genome mutagenesis. The study of purified AID/APOBEC3 proteins is challenged by difficulties with their expression and purification arising from genotoxicity in expression hosts, extensive non-specific protein-protein/DNA/RNA interactions and haphazard oligomerization. To date, expression hosts for purification of AID/APOBEC3 enzymes include bacteria, insect and mammalian cells. Here the establishment and optimization of a yeast expression/secretion system for AID/APOBEC3s are reported, followed by comparison with the same enzymes expressed in bacterial and mammalian hosts. AID and APOBEC3G were expressed successfully in Pichia pastoris, each either with an N-terminal GST tag, C-terminal V5-His tag or as untagged native form. It was verified that the yeast-expressed enzymes exhibit identical biochemical properties to those reported using bacterial and mammalian expression, indicating high fidelity of protein folding. It was demonstrated that the system can be adapted for secretion of the enzymes into the media which was used directly in various enzyme assays. The system is also amenable to elimination of bulky fusion tags, providing native untagged enzymes. Thus, P. pastoris is an advantageous expression factory for AID/APOBEC3 enzymes, considering the cost, time, efficiency and quality of the obtained enzymes. The first report is also provided here of a functionally active, untagged, secreted AID, which may become a useful research reagent. A comprehensive comparison is made of the effect of fusion tags and expression hosts on the biochemical actions of AID and APOBEC3G.


Assuntos
Desaminases APOBEC/biossíntese , Desaminases APOBEC/genética , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Imunidade , Neoplasias/enzimologia , Pichia/genética , Desaminases APOBEC/isolamento & purificação , Citidina Desaminase/isolamento & purificação , Humanos , Mutagênicos , Neoplasias/metabolismo
17.
PLoS Pathog ; 15(2): e1007533, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30768644

RESUMO

Retroviruses have evolved multiple means to counteract host restriction factors such as single-stranded DNA-specific deoxycytidine deaminases (APOBEC3s, A3s). These include exclusion of A3s from virions by an A3-unreactive nucleocapsid or expression of an A3-neutralizing protein (Vif, Bet). However, a number of retroviruses package A3s and do not encode apparent vif- or bet-like genes, yet they replicate in the presence of A3s. The mode by which they overcome deleterious restriction remains largely unknown. Here we show that the prototypic betaretrovirus, mouse mammary tumor virus (MMTV), packages similar amounts of A3s as HIV-1ΔVif, yet its proviruses carry a significantly lower level of A3-mediated deamination events than the lentivirus. The G-to-A mutation rate increases when the kinetics of reverse transcription is reduced by introducing a mutation (F120L) to the DNA polymerase domain of the MMTV reverse transcriptase (RT). A similar A3-sensitizing effect was observed when the exposure time of single-stranded DNA intermediates to A3s during reverse transcription was lengthened by reducing the dNTP concentration or by adding suboptimal concentrations of an RT inhibitor to infected cells. Thus, the MMTV RT has evolved to impede access of A3s to transiently exposed minus DNA strands during reverse transcription, thereby alleviating inhibition by A3 family members. A similar mechanism may be used by other retroviruses and retrotransposons to reduce deleterious effects of A3 proteins.


Assuntos
Citidina Desaminase/genética , Citosina Desaminase/genética , Vírus do Tumor Mamário do Camundongo/genética , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismo , Animais , Linhagem Celular , Citidina Desaminase/metabolismo , Citosina Desaminase/metabolismo , DNA , DNA de Cadeia Simples , Células HEK293 , Células HeLa , Humanos , Vírus do Tumor Mamário do Camundongo/crescimento & desenvolvimento , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Mutação/genética , Nucleocapsídeo , Polimerização , Ligação Proteica , Retroviridae , Transcrição Reversa/genética , Vírion
18.
Genes (Basel) ; 10(2)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30781903

RESUMO

Despite recent advances in N6-methyladenosine (m6A) biology, the regulation of crucial RNA processing steps by the RNA methyltransferase-like 3 (METTL3) in glioma stem-like cells (GSCs) remains obscure. An integrated analysis of m6A-RIP (RNA immunoprecipitation) and total RNA-Seq of METTL3-silenced GSCs identified that m6A modification in GSCs is principally carried out by METTL3. The m6A-modified transcripts showed higher abundance compared to non-modified transcripts. Further, we showed that the METTL3 is essential for the expression of GSC-specific actively transcribed genes. Silencing METTL3 resulted in the elevation of several aberrant alternative splicing events. We also found that putative m6A reader proteins play a key role in the RNA stabilization function of METTL3. METTL3 altered A-to-I and C-to-U RNA editing events by differentially regulating RNA editing enzymes ADAR and APOBEC3A. Similar to protein-coding genes, lincRNAs (long intergenic non-coding RNAs) with m6A marks showed METTL3-dependent high expression. m6A modification of 3'UTRs appeared to result in a conformation-dependent hindrance to miRNA binding to their targets. The integrated analysis of the m6A regulome in METTL3-silenced GSCs showed global disruption in tumorigenic pathways that are indispensable for GSC maintenance and glioma progression. We conclude that METTL3 plays a vital role in many steps of RNA processing and orchestrates successful execution of oncogenic pathways in GSCs.


Assuntos
Adenosina/análogos & derivados , Glioma/genética , Metiltransferases/genética , Transcrição Genética , Regiões 3' não Traduzidas/genética , Adenosina/genética , Adenosina Desaminase/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Histonas/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Conformação de Ácido Nucleico , Proteínas/genética , Edição de RNA/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética
19.
BMC Med Genet ; 20(1): 21, 2019 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660178

RESUMO

BACKGROUND: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression. METHODS: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool. RESULTS: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively. CONCLUSIONS: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .


Assuntos
Desaminase APOBEC-3G/genética , Aminoidrolases/genética , Citidina Desaminase/genética , Citosina Desaminase/genética , Infecções por HIV/genética , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Éxons , Feminino , Frequência do Gene , Testes Genéticos , Infecções por HIV/etnologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA , África do Sul/etnologia , Adulto Jovem
20.
Asia Pac J Clin Oncol ; 15(6): 275-287, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30693645

RESUMO

Previous studies have found inconsistent results regarding gene deletion in APOBEC3 (apolipoprotein B mRNA-editing catalytic polypeptide-like 3) and risk of cancer. We conducted a meta-analysis of all eligible case-control studies to find out the associations between APOBEC3 deletion and cancer risk by pooling the odds ratios (ORs) and corresponding 95% confidence intervals (CIs). Overall, the findings from 20 studies (13 articles) involving of a total of 26 225 cases and 37 201 controls revealed that DD genotype was associated significantly with increased cancer risk compared to II genotype (OR = 1.25, 95% CI = 1.01-1.56, P = 0.04). Stratified analysis from 10 studies including 14 757 cases and 17 930 controls revealed that I/D variant significantly increased the risk of breast cancer in heterozygous codominant (OR = 1.15, 95% CI = 1.03-1.28, P = 0.02, ID vs II), dominant (OR = 1.15, 95% CI = 1.01-1.31, P = 0.03, ID + DD vs II), overdominant (OR = 1.11, 95% CI = 1.05-1.25, P < 0.0001, ID vs DD + II) and allele (OR = 1.15, 95% CI = 1.13-1.25, P = 0.03, D vs I) inheritance models. In conclusion, the data propose that APOBEC3 deletion is significantly associated with increased susceptibility to cancer in overall and breast cancer. Our findings require well-designed replication in a larger independent genetic association study with larger sample sizes in diverse ethnicities.


Assuntos
Citidina Desaminase/genética , Predisposição Genética para Doença/genética , Neoplasias/genética , Neoplasias da Mama/genética , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Razão de Chances , Fatores de Risco , Deleção de Sequência
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