Nucleic Acids Research最新文献_第8页

Telomemore enables single-cell analysis of cell cycle and chromatin condensation

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf031

Iryna Yakovenko, Ionut Sebastian Mihai, Martin Selinger, William Rosenbaum, Andy Dernstedt, Remigius Gröning, Johan Trygg, Laura Carroll, Mattias Forsell, Johan Henriksson

{"title":"Telomemore enables single-cell analysis of cell cycle and chromatin condensation","authors":"Iryna Yakovenko, Ionut Sebastian Mihai, Martin Selinger, William Rosenbaum, Andy Dernstedt, Remigius Gröning, Johan Trygg, Laura Carroll, Mattias Forsell, Johan Henriksson","doi":"10.1093/nar/gkaf031","DOIUrl":"https://doi.org/10.1093/nar/gkaf031","url":null,"abstract":"Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"4 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143056342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An evolved, orthogonal ssDNA generator for targeted hypermutation of multiple genomic loci

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf051

Weiran Chu, Rongzhen Tian, Yaxin Guo, Yaokang Wu, Fabian B H Rehm, Long Liu, Jianghua Li, Guocheng Du, Jian Chen, Yanfeng Liu

{"title":"An evolved, orthogonal ssDNA generator for targeted hypermutation of multiple genomic loci","authors":"Weiran Chu, Rongzhen Tian, Yaxin Guo, Yaokang Wu, Fabian B H Rehm, Long Liu, Jianghua Li, Guocheng Du, Jian Chen, Yanfeng Liu","doi":"10.1093/nar/gkaf051","DOIUrl":"https://doi.org/10.1093/nar/gkaf051","url":null,"abstract":"Achieving targeted hypermutation of specific genomic sequences without affecting other regions remains a key challenge in continuous evolution. To address this, we evolved a T7 RNA polymerase (RNAP) mutant that synthesizes single-stranded DNA (ssDNA) instead of RNA in vivo, while still exclusively recognizing the T7 promoter. By increasing the error rate of the T7 RNAP mutant, it generates mutated ssDNA that recombines with homologous sequences in the genome, leading to targeted genomic hypermutation. This approach, termed T7 RNAP mutant-assisted continuous evolution (T7ACE), functions effectively in both typical prokaryotic and eukaryotic microorganisms (Escherichia coli and Saccharomyces cerevisiae), achieving targeted hypermutations at rates 2800- and 1200-fold higher than the genomic mutation rates, respectively. Using T7ACE, we successfully evolved an eight-fold increase in tigecycline resistance within 7 days and doubled the efficiency of a xylose utilization pathway in 10 days, demonstrating the efficiency and broad applicability of this single-component tool for continuous evolution.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"36 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf027

Lei Zhang, Qiuxian Yang, Yuekun Shao, Shenyang Ding, Jiamei Guo, George F Gao, Tao Deng

{"title":"Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch","authors":"Lei Zhang, Qiuxian Yang, Yuekun Shao, Shenyang Ding, Jiamei Guo, George F Gao, Tao Deng","doi":"10.1093/nar/gkaf027","DOIUrl":"https://doi.org/10.1093/nar/gkaf027","url":null,"abstract":"The heterotrimeric RNA-dependent RNA polymerase (RdRp) of influenza A virus catalyzes viral RNA transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) by adopting different conformations. A switch from transcription to replication occurs at a relatively late stage of infection. We recently reported that the viral NS2 protein, expressed at later stages from a spliced transcript of the NS segment messenger RNA (mRNA), inhibits transcription, promotes replication and plays a key role in the transcription-to-replication switch. In this study, we performed comprehensive functional analyses to elucidate how NS2 promotes viral genome replication. Using a cell-based single-step RNP reconstitution assay, we found that NS2 specifically promotes the first-step vRNA-to-cRNA synthesis. Further investigation revealed that this promotion is tightly associated with the intrinsic properties of the 3′-vRNA promoter. Employing a highly sensitive complementation reporter assay, we demonstrated that NS2 associates more strongly with the vRNA-resident RdRp than the cRNA-resident RdRp. These findings were further validated through in vitro replication analyses. We, therefore, propose that influenza A virus NS2 protein targets vRNA-resident RdRp to drive the transcription-to-replication switch during infection.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"60 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

WBSCR16 is essential for mitochondrial 16S rRNA processing in mammals

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkae1325

Shengjie Zhang, Zi Dong, Yang Feng, Wei Guo, Chen Zhang, Yifan Shi, Zhiyun Zhao, Jiqiu Wang, Guang Ning, Guorui Huang

{"title":"WBSCR16 is essential for mitochondrial 16S rRNA processing in mammals","authors":"Shengjie Zhang, Zi Dong, Yang Feng, Wei Guo, Chen Zhang, Yifan Shi, Zhiyun Zhao, Jiqiu Wang, Guang Ning, Guorui Huang","doi":"10.1093/nar/gkae1325","DOIUrl":"https://doi.org/10.1093/nar/gkae1325","url":null,"abstract":"Mitochondrial rRNAs play important roles in regulating mtDNA-encoded gene expression and energy metabolism subsequently. However, the proteins that regulate mitochondrial 16S rRNA processing remain poorly understood. Herein, we generated adipose-specific Wbscr16-/-mice and cells, both of which exhibited dramatic mitochondrial changes. Subsequently, WBSCR16 was identified as a 16S rRNA-binding protein essential for the cleavage of 16S rRNA-mt-tRNALeu, facilitating 16S rRNA processing and mitochondrial ribosome assembly. Additionally, WBSCR16 recruited RNase P subunit MRPP3 to nascent 16S rRNA and assisted in this specific cleavage. Furthermore, evidence showed that adipose-specific Wbscr16 ablation promotes energy wasting via lipid preference in brown adipose tissue, leading to excess energy expenditure and resistance to obesity. In contrast, overexpression of WBSCR16 upregulated 16S rRNA processing and induced a preference for glucose utilization in both transgenic mouse models and cultured cells. These findings suggest that WBSCR16 plays essential roles in mitochondrial 16S rRNA processing in mammals, and is the key mitochondrial protein to balance glucose and lipid metabolism.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"40 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143056340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

The ortholog of human DNAJC9 promotes histone H3–H4 degradation and is counteracted by Asf1 in fission yeast

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf036

Yan Ding, Jun Li, He-Li Jiang, Fang Suo, Guang-Can Shao, Xiao-Ran Zhang, Meng-Qiu Dong, Chao-Pei Liu, Rui-Ming Xu, Li-Lin Du

{"title":"The ortholog of human DNAJC9 promotes histone H3–H4 degradation and is counteracted by Asf1 in fission yeast","authors":"Yan Ding, Jun Li, He-Li Jiang, Fang Suo, Guang-Can Shao, Xiao-Ran Zhang, Meng-Qiu Dong, Chao-Pei Liu, Rui-Ming Xu, Li-Lin Du","doi":"10.1093/nar/gkaf036","DOIUrl":"https://doi.org/10.1093/nar/gkaf036","url":null,"abstract":"Mammalian J-domain protein DNAJC9 interacts with histones H3–H4 and is important for cell proliferation. However, its exact function remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, loss of Djc9, the ortholog of DNAJC9, renders the histone chaperone Asf1 no longer essential for growth. Utilizing AlphaFold-based structural prediction, we identified a histone-binding surface on Djc9 that binds to helix α3 of H3 in a manner that precludes simultaneous helix α3-binding by Asf1. Djc9 and Asf1 indeed compete for binding to the H3–H4 dimer in vitro, and an H3-α3 mutation impeding Djc9 binding also renders Asf1 non-essential, indicating that the role of Asf1 needed for growth in fission yeast is to prevent histone binding by Djc9. In the absence of Asf1, cell growth is hindered due to unrestrained Djc9-mediated downregulation of H3 and H4. In the presence of Asf1, Djc9 confers resistance to the DNA replication inhibitor hydroxyurea and dominant negative disease-related histone mutants by promoting the degradation of superfluous or dysfunctional histones. Our findings provide new insights into the function and mechanism of this conserved histone-binding protein.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"26 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143056338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Measuring XNA polymerase fidelity in a hydrogel particle format

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf038

Esau L Medina, John C Chaput

{"title":"Measuring XNA polymerase fidelity in a hydrogel particle format","authors":"Esau L Medina, John C Chaput","doi":"10.1093/nar/gkaf038","DOIUrl":"https://doi.org/10.1093/nar/gkaf038","url":null,"abstract":"Growth in the development of engineered polymerases for synthetic biology has led to renewed interest in assays that can measure the fidelity of polymerases that are capable of synthesizing artificial genetic polymers (XNAs). Conventional approaches require purifying the XNA intermediate of a replication cycle (DNA → XNA → DNA) by denaturing polyacrylamide gel electrophoresis, which is a slow, costly, and inefficient process that requires a large-scale transcription reaction and careful extraction of the XNA strand from the gel slice. In an effort to streamline the assay, we developed a purification-free approach in which the XNA transcription and reverse transcription steps occur inside the matrix of a hydrogel-coated magnetic particle. Accordingly, a DNA primer cross-linked throughout the gel matrix is annealed to a template of defined sequence and extended with XNA. Following removal of the DNA template, the XNA product strand is copied back into DNA, recovered, amplified, cloned, and sequenced. Performing the replication cycle in the hydrogel format drastically reduces the time and reaction scales required to measure the fidelity of an XNA polymerase, making it easier to evaluate the properties of a range of candidate XNA polymerases.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"593 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143056339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Selection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf021

Muhoon Lee, Taisei Wakigawa, Qimin Jia, Chang Liu, Ruiyuan Huang, Shuai Huang, Asuteka Nagao, Tsutomu Suzuki, Kozo Tomita, Shintaro Iwasaki, Nono Takeuchi-Tomita

{"title":"Selection of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation","authors":"Muhoon Lee, Taisei Wakigawa, Qimin Jia, Chang Liu, Ruiyuan Huang, Shuai Huang, Asuteka Nagao, Tsutomu Suzuki, Kozo Tomita, Shintaro Iwasaki, Nono Takeuchi-Tomita","doi":"10.1093/nar/gkaf021","DOIUrl":"https://doi.org/10.1093/nar/gkaf021","url":null,"abstract":"The mammalian mitochondrial protein synthesis system produces 13 essential subunits of oxidative phosphorylation (OXPHOS) complexes. Translation initiation in mammalian mitochondria is characterized by the use of leaderless messenger RNAs (mRNAs) and non-AUG start codons, where the proofreading function of IF-3mt still remains elusive. Here, we developed a reconstituted mammalian mitochondrial translation system using in vitro transcribed and native mitochondrial transfer RNAs (tRNAs) to investigate IF-3mt’s proofreading function. Similar to bacterial IF-3, IF-3mt permits an initiator tRNA to participate in initiation by discriminating the three G–C pairs in its anticodon stem, and by the cognate interactions of its anticodon with the AUG start codon. As a result, IF-3mt promotes the accurate initiation of leaderless mRNAs. Nevertheless, IF-3mt can also facilitate initiation from the non-AUG(AUA) start codon through its unique N- and C-terminal extensions, in concert with the 5-methylcytidine (m5C) or 5-formylcytidine (f5C) modification at the anticodon wobble position of mt-tRNAMet. This is partly because the IF-3mt-specific N- and C-terminal extensions and the KKGK-motif favor leaderless mRNA initiation and relax non-AUG start codon discrimination. Analyses of IF-3mt-depleted human cells revealed that IF-3mt indeed participates in translating the open reading frames (ORFs) of leaderless mRNAs, as well as the internal ORFs of dicistronic mRNAs.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"29 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143056337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel ADP-directed chaperone function facilitates the ATP-driven motor activity of SARS-CoV helicase

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-29 DOI: 10.1093/nar/gkaf034

Jeongmin Yu, Hyeryeon Im, HyeokJin Cho, Yongmoon Jeon, Jong-Bong Lee, Gwangrog Lee

{"title":"A novel ADP-directed chaperone function facilitates the ATP-driven motor activity of SARS-CoV helicase","authors":"Jeongmin Yu, Hyeryeon Im, HyeokJin Cho, Yongmoon Jeon, Jong-Bong Lee, Gwangrog Lee","doi":"10.1093/nar/gkaf034","DOIUrl":"https://doi.org/10.1093/nar/gkaf034","url":null,"abstract":"Helicase is a nucleic acid motor that catalyses the unwinding of double-stranded (ds) RNA and DNA via ATP hydrolysis. Helicases can act either as a nucleic acid motor that unwinds its ds substrates or as a chaperone that alters the stability of its substrates, but the two activities have not yet been reported to act simultaneously. Here, we used single-molecule techniques to unravel the synergistic coordination of helicase and chaperone activities, and found that the severe acute respiratory syndrome coronavirus helicase (nsp13) is capable of two modes of action: (i) binding of nsp13 in tandem with the fork junction of the substrate mechanically unwinds the substrate by an ATP-driven synchronous power stroke; and (ii) free nsp13, which is not bound to the substrate but complexed with ADP in solution, destabilizes the substrate through collisions between transient binding and unbinding events with unprecedented melting capability. Our findings provide new insights into how the same enzyme works via two modes on different parts of the substrate and synergistically catalyses the unwinding reaction, utilizing ATP and recycling its by-product ADP as an energy source.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"74 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at super-enhancers

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-25 DOI: 10.1093/nar/gkaf015

Shengyuan Wang, Zhenjia Wang, Chongzhi Zang

{"title":"Genomic clustering tendency of transcription factors reflects phase-separated transcriptional condensates at super-enhancers","authors":"Shengyuan Wang, Zhenjia Wang, Chongzhi Zang","doi":"10.1093/nar/gkaf015","DOIUrl":"https://doi.org/10.1093/nar/gkaf015","url":null,"abstract":"Many transcription factors (TFs) have been shown to bind to super-enhancers, forming transcriptional condensates to activate transcription in various cellular systems. However, the genomic and epigenomic determinants of phase-separated transcriptional condensate formation remain poorly understood. Questions regarding which TFs tend to associate with transcriptional condensates and what factors influence their association are largely unanswered. Here we systematically analyzed 571 DNA sequence motifs across the human genome and 6650 TF binding profiles across different cell types to identify the molecular features contributing to the formation of transcriptional condensates. We found that the genomic distributions of sequence motifs for different TFs exhibit distinct clustering tendencies. Notably, TF motifs with a high genomic clustering tendency are significantly associated with super-enhancers. TF binding profiles showing a high genomic clustering tendency are further enriched at cell-type-specific super-enhancers. TFs with a high binding clustering tendency also possess high liquid–liquid phase separation abilities. Compared to nonclustered TF binding, densely clustered TF binding sites are more enriched at cell-type-specific super-enhancers with higher chromatin accessibility, elevated chromatin interaction and stronger association with cancer outcomes. Our results indicate that the clustered genomic binding patterns and the phase separation properties of TFs collectively contribute to the formation of transcriptional condensates.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"63 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143035145","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

AAV vector transduction restriction and attenuated toxicity in hESCs via a rationally designed inverted terminal repeat

IF 14.9 2区生物学

Nucleic Acids Research Pub Date : 2025-01-25 DOI: 10.1093/nar/gkaf013

Liujiang Song, Tomoko Hasegawa, Nolan J Brown, Jacquelyn J Bower, Richard J Samulski, Matthew L Hirsch

{"title":"AAV vector transduction restriction and attenuated toxicity in hESCs via a rationally designed inverted terminal repeat","authors":"Liujiang Song, Tomoko Hasegawa, Nolan J Brown, Jacquelyn J Bower, Richard J Samulski, Matthew L Hirsch","doi":"10.1093/nar/gkaf013","DOIUrl":"https://doi.org/10.1093/nar/gkaf013","url":null,"abstract":"Adeno-associated virus (AAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a synthetic ITR (SynITR), harboring substitutions in putative p53 binding sites was generated and evaluated for vector production and gene delivery. Replication of SynITR flanked transgenic genome was similar compared to wild type (wt) ITR, with a modest increase in vector titers. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cells without toxicity. Following AAV2-wtITR vector infection of hESCs, rapid apoptosis was observed as reported. In contrast, infection by AAV2 vectors packaged with SynITRs attenuated the wtITR-induced hESC toxicity. While hESC particle entry and the abundance of double stranded circular episomes was similar for the ITR contexts, reporter expression was inhibited from transduced SynITR genomes. Mechanistically, infection of hESCs induced γH2AX in an ITR-independent manner, however, canonical activation of p53α was uncoupled using AAV-SynITR. Further investigations in hESCs revealed additional novel findings: (i) p53β is uniquely and constitutively active and (ii) AAV vector infection, independent of the ITR sequence, induces activation of p53ψ. The data herein reveal an ITR-dependent AAV vector transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"58 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143035143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0