RESUMO
During each cell cycle, the process of DNA replication timing is tightly regulated to ensure the accurate duplication of the genome. The extent and significance of alterations in this process during malignant transformation have not been extensively explored. Here, we assess the impact of altered replication timing (ART) on cancer evolution by analysing replication-timing sequencing of cancer and normal cell lines and 952 whole-genome sequenced lung and breast tumours. We find that 6%-18% of the cancer genome exhibits ART, with regions with a change from early to late replication displaying an increased mutation rate and distinct mutational signatures. Whereas regions changing from late to early replication contain genes with increased expression and present a preponderance of APOBEC3-mediated mutation clusters and associated driver mutations. We demonstrate that ART occurs relatively early during cancer evolution and that ART may have a stronger correlation with mutation acquisition than alterations in chromatin structure.
Assuntos
Neoplasias da Mama , Período de Replicação do DNA , Neoplasias Pulmonares , Mutação , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Linhagem Celular Tumoral , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Taxa de Mutação , Replicação do DNA/genética , Genoma HumanoRESUMO
The APOBEC/AID family is known for its mutator activity, and recent evidence also supports the potential impact of ADARs. Furthermore, the mutator impacts of APOBEC/ADAR mutations have not yet been investigated. Assessment of pancancer TCGA exomes identified enriched somatic variants among exomes with nonsynonymous APOBEC1, APOBEC3B, APOBEC3C, ADAR, and ADARB1 mutations, compared to exomes with synonymous ones. Principal component (PC) analysis reduced the number of potential players to eight in cancer exomes/genomes, and to five in cancer types. Multivariate regression analysis was used to assess the impact of the PCs on each COSMIC mutational signature among pancancer exomes/genomes and particular cancers, identifying several novel links, including SBS17b, SBS18, and ID7 mainly determined by APOBEC1 mRNA levels; SBS40, ID1, and ID2 by age; SBS3 and SBS16 by APOBEC3A/APOBEC3B mRNA levels; ID5 and DBS9 by DNA repair/replication (DRR) defects; and SBS7a-d, SBS38, ID4, ID8, ID13, and DBS1 by ultraviolet (UV) radiation/ADARB1 mRNA levels. APOBEC/ADAR mutations appeared to potentiate the impact of DRR defects on several mutational signatures, and some factors seemed to inversely affect certain signatures. These findings potentially implicate certain APOBEC/ADAR mutations/mRNA levels in distinct mutational signatures, particularly APOBEC1 mRNA levels in aging-related signatures and ADARB1 mRNA levels in UV radiation-related signatures.
Assuntos
Adenosina Desaminase , Envelhecimento , Mutação , RNA Mensageiro , Proteínas de Ligação a RNA , Raios Ultravioleta , Humanos , Raios Ultravioleta/efeitos adversos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Envelhecimento/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Neoplasias/genética , ExomaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.
Assuntos
Desaminases APOBEC , COVID-19 , Citidina Desaminase , SARS-CoV-2 , Replicação Viral , Humanos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , SARS-CoV-2/metabolismo , Desaminases APOBEC/metabolismo , Desaminases APOBEC/genética , COVID-19/virologia , COVID-19/metabolismo , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Células THP-1 , Mutação , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Genoma ViralRESUMO
Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.
Assuntos
Pareamento Incorreto de Bases , Dano ao DNA , DNA de Cadeia Simples , Análise de Sequência de DNA , Imagem Individual de Molécula , Humanos , Envelhecimento/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Pareamento Incorreto de Bases/genética , Citidina Desaminase/metabolismo , Citidina Desaminase/genética , Citosina/metabolismo , Desaminação , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Genoma Mitocondrial/genética , Mutação , Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Imagem Individual de Molécula/métodos , Masculino , FemininoAssuntos
Citidina Desaminase , Desoxicitidina , Resistencia a Medicamentos Antineoplásicos , Gencitabina , Regulação para Cima , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Regulação para Cima/efeitos dos fármacos , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular TumoralRESUMO
The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.
Assuntos
Cromatina , Proteínas Musculares , Desaminases APOBEC/genética , Desaminase APOBEC-1/genética , Diferenciação Celular/genética , Cromatina/genética , Citidina Desaminase/metabolismo , DNA , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos/metabolismo , RNA Mensageiro/genética , Animais , CamundongosRESUMO
In mammals, RNA editing events involve the conversion of adenosine (A) in inosine (I) by ADAR enzymes or the hydrolytic deamination of cytosine (C) in uracil (U) by the APOBEC family of enzymes, mostly APOBEC1. RNA editing has a plethora of biological functions, and its deregulation has been associated with various human disorders. While the large-scale detection of A-to-I is quite straightforward using the Illumina RNAseq technology, the identification of C-to-U events is a non-trivial task. This difficulty arises from the rarity of such events in eukaryotic genomes and the challenge of distinguishing them from background noise. Direct RNA sequencing by Oxford Nanopore Technology (ONT) permits the direct detection of Us on sequenced RNA reads. Surprisingly, using ONT reads from wild-type (WT) and APOBEC1-knock-out (KO) murine cell lines as well as in vitro synthesized RNA without any modification, we identified a systematic error affecting the accuracy of the Cs call, thereby leading to incorrect identifications of C-to-U events. To overcome this issue in direct RNA reads, here we introduce a novel machine learning strategy based on the isolation Forest (iForest) algorithm in which C-to-U editing events are considered as sequencing anomalies. Using in vitro synthesized and human ONT reads, our model optimizes the signal-to-noise ratio improving the detection of C-to-U editing sites with high accuracy, over 90% in all samples tested. Our results suggest that iForest, known for its rapid implementation and minimal memory requirements, is a promising tool to denoise ONT reads and reliably identify RNA modifications.
Assuntos
Edição de RNA , RNA , Camundongos , Animais , Humanos , RNA/genética , Sequência de Bases , Desaminases APOBEC/genética , Mamíferos/genética , Análise de Sequência de RNARESUMO
Since November 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the worldwide pandemic of the coronavirus disease 2019 (COVID-19), the impact of which is huge to the lives of world populations. Many studies suggested that such situation will continue due to the endless mutations in SARS-CoV-2 genome that result in complexity of the efforts for the control of SARS-CoV-2, since the special enrichment of nucleotide substitution C>U in SARS-CoV-2 sequences were discovered mainly due to the editing by human host factors APOBEC3 genes. The observation of SARS-CoV-2 variants Beta (B.1.351) and Omicron (B.1.1.529) firstly spreading in South Africa promoted us to hypothesize that genetic variants of APOBEC3 special in African populations may be attributed to the higher mutation rate of SARS-CoV-2 variants in Africa. Current study was conducted to search for functional variants of APOBEC3 genes associate with COVID-19 hospitalization in African population. By integrating data from the 1000 Genomes Project, Genotype-Tissue Expression (GTEx), and Host Genetics Initiative (HGI) of COVID-19, we identified potential functional SNPs close to APOBEC3 genes that are associated with COVID-19 hospitalization in African but not with other populations. Our study provides new insights on the potential contribution of APOBEC3 genes on the evolution of SARS-CoV-2 mutations in African population, but further replication is needed to confirm our results.