Ionizing radiation induces various types of DNA damage, and the reparability and lethal effects of DNA damage differ depending on its spatial density. Elucidating the structure of radiation-induced clustered DNA damage and its repair processes will enhance our understanding of the lethal impact of ionizing radiation and advance progress toward precise therapeutics. Previously, we developed a method to directly visualize DNA damage using atomic force microscopy (AFM) and classified clustered DNA damage into simple base damage clusters (BDCs), complex BDCs and complex double-strand breaks (DSBs). This study investigated the repair of each type of damage in DNA-repair-deficient human TK6 cells and elucidated the association between each type of clustered DNA damage and the pathway responsible for its repair postirradiation with low linear energy transfer (LET) radiation (X-rays) and high-LET radiation (Fe-ion beams) in cells. We found that base excision repair and, surprisingly, nucleotide excision repair restored simple and complex BDCs. In addition, the number of complex DSBs in wild-type cells increases 1 h postirradiation, which was most likely caused by BDC cleavage initiated with DNA glycosylases. Furthermore, complex DSBs, which are likely associated with lethality, are repaired by homologous recombination with little contribution from nonhomologous-end joining.
{"title":"Deciphering repair pathways of clustered DNA damage in human TK6 cells: insights from atomic force microscopy direct visualization","authors":"Toshiaki Nakano, Ken Akamatsu, Masaoki Kohzaki, Masataka Tsuda, Ryoichi Hirayama, Akira Sassa, Manabu Yasui, Mahmoud I Shoulkamy, Takeshi Hiromoto, Taro Tamada, Hiroshi Ide, Naoya Shikazono","doi":"10.1093/nar/gkae1077","DOIUrl":"https://doi.org/10.1093/nar/gkae1077","url":null,"abstract":"Ionizing radiation induces various types of DNA damage, and the reparability and lethal effects of DNA damage differ depending on its spatial density. Elucidating the structure of radiation-induced clustered DNA damage and its repair processes will enhance our understanding of the lethal impact of ionizing radiation and advance progress toward precise therapeutics. Previously, we developed a method to directly visualize DNA damage using atomic force microscopy (AFM) and classified clustered DNA damage into simple base damage clusters (BDCs), complex BDCs and complex double-strand breaks (DSBs). This study investigated the repair of each type of damage in DNA-repair-deficient human TK6 cells and elucidated the association between each type of clustered DNA damage and the pathway responsible for its repair postirradiation with low linear energy transfer (LET) radiation (X-rays) and high-LET radiation (Fe-ion beams) in cells. We found that base excision repair and, surprisingly, nucleotide excision repair restored simple and complex BDCs. In addition, the number of complex DSBs in wild-type cells increases 1 h postirradiation, which was most likely caused by BDC cleavage initiated with DNA glycosylases. Furthermore, complex DSBs, which are likely associated with lethality, are repaired by homologous recombination with little contribution from nonhomologous-end joining.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"87 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142962801","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}
Simonas Juzenas, Karolis Goda, Vaidotas Kiseliovas, Justina Zvirblyte, Alvaro Quintinal-Villalonga, Juozas Siurkus, Juozas Nainys, Linas Mazutis
The expansion of single-cell analytical techniques has empowered the exploration of diverse biological questions at the individual cells. Droplet-based single-cell RNA sequencing (scRNA-seq) methods have been particularly widely used due to their high-throughput capabilities and small reaction volumes. While commercial systems have contributed to the widespread adoption of droplet-based scRNA-seq, their relatively high cost limits the ability to profile large numbers of cells and samples. Moreover, as the scale of single-cell sequencing continues to expand, accommodating diverse workflows and cost-effective multi-biospecimen profiling becomes more critical. Herein, we present inDrops-2, an open-source scRNA-seq technology designed to profile live or preserved cells with a sensitivity matching that of state-of-the-art commercial systems but at a 6-fold lower cost. We demonstrate the flexibility of inDrops-2, by implementing two prominent scRNA-seq protocols, based on exponential and linear amplification of barcoded-complementary DNA, and provide useful insights into the advantages and disadvantages inherent to each approach. We applied inDrops-2 to simultaneously profile multiple human lung carcinoma samples that had been subjected to cell preservation, long-term storage and multiplexing to obtain a multiregional cellular profile of the tumor microenvironment. The scalability, sensitivity and cost efficiency make inDrops-2 stand out among other droplet-based scRNA-seq methods, ideal for large-scale studies on rare cell molecular signatures.
{"title":"inDrops-2: a flexible, versatile and cost-efficient droplet microfluidic approach for high-throughput scRNA-seq of fresh and preserved clinical samples","authors":"Simonas Juzenas, Karolis Goda, Vaidotas Kiseliovas, Justina Zvirblyte, Alvaro Quintinal-Villalonga, Juozas Siurkus, Juozas Nainys, Linas Mazutis","doi":"10.1093/nar/gkae1312","DOIUrl":"https://doi.org/10.1093/nar/gkae1312","url":null,"abstract":"The expansion of single-cell analytical techniques has empowered the exploration of diverse biological questions at the individual cells. Droplet-based single-cell RNA sequencing (scRNA-seq) methods have been particularly widely used due to their high-throughput capabilities and small reaction volumes. While commercial systems have contributed to the widespread adoption of droplet-based scRNA-seq, their relatively high cost limits the ability to profile large numbers of cells and samples. Moreover, as the scale of single-cell sequencing continues to expand, accommodating diverse workflows and cost-effective multi-biospecimen profiling becomes more critical. Herein, we present inDrops-2, an open-source scRNA-seq technology designed to profile live or preserved cells with a sensitivity matching that of state-of-the-art commercial systems but at a 6-fold lower cost. We demonstrate the flexibility of inDrops-2, by implementing two prominent scRNA-seq protocols, based on exponential and linear amplification of barcoded-complementary DNA, and provide useful insights into the advantages and disadvantages inherent to each approach. We applied inDrops-2 to simultaneously profile multiple human lung carcinoma samples that had been subjected to cell preservation, long-term storage and multiplexing to obtain a multiregional cellular profile of the tumor microenvironment. The scalability, sensitivity and cost efficiency make inDrops-2 stand out among other droplet-based scRNA-seq methods, ideal for large-scale studies on rare cell molecular signatures.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"57 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142961649","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}
Progress in biology has generated numerous lists of genes that share some property. But advancing from these lists of genes to understanding their roles is slow and unsystematic. Here we use RNA silencing in Caenorhabditis elegans to illustrate an approach for prioritizing genes for detailed study given limited resources. The partially subjective relationships between genes forged by both deduced functional relatedness and biased progress in the field were captured as mutual information and used to cluster genes that were frequently identified yet remain understudied. Some proteins encoded by these understudied genes are predicted to physically interact with known regulators of RNA silencing, suggesting feedback regulation. Predicted interactions with proteins that act in other processes and the clustering of studied genes among the most frequently perturbed suggest regulatory links connecting RNA silencing to other processes like the cell cycle and asymmetric cell division. Thus, among the gene products altered when a process is perturbed could be regulators of that process acting to restore homeostasis, which provides a way to use RNA sequencing to identify candidate protein–protein interactions. Together, the analysis of perturbed transcripts and potential interactions of the proteins they encode could help prioritize candidate regulators of any process.
{"title":"Selecting genes for analysis using historically contingent progress: from RNA changes to protein–protein interactions","authors":"Farhaan Lalit, Antony M Jose","doi":"10.1093/nar/gkae1246","DOIUrl":"https://doi.org/10.1093/nar/gkae1246","url":null,"abstract":"Progress in biology has generated numerous lists of genes that share some property. But advancing from these lists of genes to understanding their roles is slow and unsystematic. Here we use RNA silencing in Caenorhabditis elegans to illustrate an approach for prioritizing genes for detailed study given limited resources. The partially subjective relationships between genes forged by both deduced functional relatedness and biased progress in the field were captured as mutual information and used to cluster genes that were frequently identified yet remain understudied. Some proteins encoded by these understudied genes are predicted to physically interact with known regulators of RNA silencing, suggesting feedback regulation. Predicted interactions with proteins that act in other processes and the clustering of studied genes among the most frequently perturbed suggest regulatory links connecting RNA silencing to other processes like the cell cycle and asymmetric cell division. Thus, among the gene products altered when a process is perturbed could be regulators of that process acting to restore homeostasis, which provides a way to use RNA sequencing to identify candidate protein–protein interactions. Together, the analysis of perturbed transcripts and potential interactions of the proteins they encode could help prioritize candidate regulators of any process.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"26 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142939690","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}
Marcos Iuri Roos Kulmann, Eliska Taborska, Brigita Benköova, Martin Palus, Ales Drobek, Filip Horvat, Josef Pasulka, Radek Malik, Eva Salyova, Vaclav Hönig, Michaela Pellerova, Maria Borsanyiova, Lenka Nedvedova, Ondrej Stepanek, Shubhada Bopegamage, Daniel Ruzek, Petr Svoboda
In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.
{"title":"Enhanced RNAi does not provide efficient innate antiviral immunity in mice","authors":"Marcos Iuri Roos Kulmann, Eliska Taborska, Brigita Benköova, Martin Palus, Ales Drobek, Filip Horvat, Josef Pasulka, Radek Malik, Eva Salyova, Vaclav Hönig, Michaela Pellerova, Maria Borsanyiova, Lenka Nedvedova, Ondrej Stepanek, Shubhada Bopegamage, Daniel Ruzek, Petr Svoboda","doi":"10.1093/nar/gkae1288","DOIUrl":"https://doi.org/10.1093/nar/gkae1288","url":null,"abstract":"In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"132 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142936960","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}
Youri Timsit, Grégoire Sergeant-Perthuis, Daniel Bennequin
Accurate protein synthesis requires ribosomes to integrate signals from distant functional sites and execute complex dynamics. Despite advances in understanding ribosome structure and function, two key questions remain: how information is transmitted between these distant sites, and how ribosomal movements are synchronized? We recently highlighted the existence of ribosomal protein networks, likely evolved to participate in ribosome signaling. Here, we investigate the relationship between ribosomal protein networks and ribosome dynamics. Our findings show that major motion centers in the bacterial ribosome interact specifically with r-proteins, and that ribosomal RNA exhibits high mobility around each r-protein. This suggests that periodic electrostatic changes in the context of negatively charged residues (Glu and Asp) induce RNA–protein ‘distance-approach’ cycles, controlling key ribosomal movements during translocation. These charged residues play a critical role in modulating electrostatic repulsion between RNA and proteins, thus coordinating ribosomal dynamics. We propose that r-protein networks synchronize ribosomal dynamics through an ‘electrostatic domino’ effect, extending the concept of allostery to the regulation of movements within supramolecular assemblies.
{"title":"The role of ribosomal protein networks in ribosome dynamics","authors":"Youri Timsit, Grégoire Sergeant-Perthuis, Daniel Bennequin","doi":"10.1093/nar/gkae1308","DOIUrl":"https://doi.org/10.1093/nar/gkae1308","url":null,"abstract":"Accurate protein synthesis requires ribosomes to integrate signals from distant functional sites and execute complex dynamics. Despite advances in understanding ribosome structure and function, two key questions remain: how information is transmitted between these distant sites, and how ribosomal movements are synchronized? We recently highlighted the existence of ribosomal protein networks, likely evolved to participate in ribosome signaling. Here, we investigate the relationship between ribosomal protein networks and ribosome dynamics. Our findings show that major motion centers in the bacterial ribosome interact specifically with r-proteins, and that ribosomal RNA exhibits high mobility around each r-protein. This suggests that periodic electrostatic changes in the context of negatively charged residues (Glu and Asp) induce RNA–protein ‘distance-approach’ cycles, controlling key ribosomal movements during translocation. These charged residues play a critical role in modulating electrostatic repulsion between RNA and proteins, thus coordinating ribosomal dynamics. We propose that r-protein networks synchronize ribosomal dynamics through an ‘electrostatic domino’ effect, extending the concept of allostery to the regulation of movements within supramolecular assemblies.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"24 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142937673","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}
Jun Kim, Jong Lyul Park, Jin Ok Yang, Sangok Kim, Soobok Joe, Gunwoo Park, Taeyeon Hwang, Mun-Jeong Cho, Seungjae Lee, Jong-Eun Lee, Ji-Hwan Park, Min-Kyung Yeo, Seon-Young Kim
Given the presence of highly repetitive genomic regions such as subtelomeric regions, understanding human genomic evolution remains challenging. Recently, long-read sequencing technology has facilitated the identification of complex genetic variants, including structural variants (SVs), at the single-nucleotide level. Here, we resolved SVs and their underlying DNA damage–repair mechanisms in subtelomeric regions, which are among the most uncharted genomic regions. We generated ∼20 × high-fidelity long-read sequencing data from three Korean individuals and their partially phased high-quality de novo genome assemblies (contig N50: 6.3–58.2 Mb). We identified 131 138 deletion and 121 461 insertion SVs, 41.6% of which were prevalent in the East Asian population. The commonality of the SVs identified among the Korean population was examined by short-read sequencing data from 103 Korean individuals, providing the first comprehensive SV set representing the population based on the long-read assemblies. Manual investigation of 19 large subtelomeric SVs (≥5 kb) and their associated repair signatures revealed the potential repair mechanisms leading to the formation of these SVs. Our study provides mechanistic insight into human telomere evolution and can facilitate our understanding of human SV formation.
{"title":"Highly accurate Korean draft genomes reveal structural variation highlighting human telomere evolution","authors":"Jun Kim, Jong Lyul Park, Jin Ok Yang, Sangok Kim, Soobok Joe, Gunwoo Park, Taeyeon Hwang, Mun-Jeong Cho, Seungjae Lee, Jong-Eun Lee, Ji-Hwan Park, Min-Kyung Yeo, Seon-Young Kim","doi":"10.1093/nar/gkae1294","DOIUrl":"https://doi.org/10.1093/nar/gkae1294","url":null,"abstract":"Given the presence of highly repetitive genomic regions such as subtelomeric regions, understanding human genomic evolution remains challenging. Recently, long-read sequencing technology has facilitated the identification of complex genetic variants, including structural variants (SVs), at the single-nucleotide level. Here, we resolved SVs and their underlying DNA damage–repair mechanisms in subtelomeric regions, which are among the most uncharted genomic regions. We generated ∼20 × high-fidelity long-read sequencing data from three Korean individuals and their partially phased high-quality de novo genome assemblies (contig N50: 6.3–58.2 Mb). We identified 131 138 deletion and 121 461 insertion SVs, 41.6% of which were prevalent in the East Asian population. The commonality of the SVs identified among the Korean population was examined by short-read sequencing data from 103 Korean individuals, providing the first comprehensive SV set representing the population based on the long-read assemblies. Manual investigation of 19 large subtelomeric SVs (≥5 kb) and their associated repair signatures revealed the potential repair mechanisms leading to the formation of these SVs. Our study provides mechanistic insight into human telomere evolution and can facilitate our understanding of human SV formation.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"1 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142936962","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}
Yunfan Xu, Shuopeng Qi, Gongrui Zhang, Dan Liu, Dejin Xu, Tong Qin, Qin Cheng, Han Kang, Bei Hu, Zhen Huang
Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments. We observed that the denatured dsDNA formed a long hybrid duplex with these mRNA fragments, overcoming their structures. Further, our novel strategy remarkably facilitated the ligation of long mRNA fragments (especially structured ones), offering ligation efficiency up to 106-fold higher than the ssDNA method. Using this one-pot strategy, we conveniently synthesized the mRNAs with N1-methylpseudouridine (m1ψ) and 5-methylcytidine (m5C) modifications in specific regions. We have found that compared with the fully modified mRNAs, the 3′UTR m1ψ modifications alone increased the translation efficiency, and the combined modifications of the m1ψ-3′UTR and m5C-5′UTR/CDS exhibited higher translation efficiency and lower immunogenicity in general. Our study presents a broadly applicable strategy for producing regionally modified mRNAs, advancing the potential of mRNA therapeutics.
{"title":"One-pot ligation of multiple mRNA fragments on dsDNA splint advancing regional modification and translation","authors":"Yunfan Xu, Shuopeng Qi, Gongrui Zhang, Dan Liu, Dejin Xu, Tong Qin, Qin Cheng, Han Kang, Bei Hu, Zhen Huang","doi":"10.1093/nar/gkae1280","DOIUrl":"https://doi.org/10.1093/nar/gkae1280","url":null,"abstract":"Region-specific RNA modifications are crucial for advancing RNA research and therapeutics, including messenger RNA (mRNA)-based vaccines and immunotherapy. However, the predominant method, synthesizing regionally modified mRNAs with short single-stranded DNA (ssDNA) splints, encounters challenges in ligating long mRNA fragments due to the formation of RNA self-folded complex structures. To address this issue, we developed an efficient strategy using an easily obtained long double-stranded DNA (dsDNA) as a ligation splint after in situ denaturing, while parts of this dsDNA are the templates for transcribing mRNA fragments. We observed that the denatured dsDNA formed a long hybrid duplex with these mRNA fragments, overcoming their structures. Further, our novel strategy remarkably facilitated the ligation of long mRNA fragments (especially structured ones), offering ligation efficiency up to 106-fold higher than the ssDNA method. Using this one-pot strategy, we conveniently synthesized the mRNAs with N1-methylpseudouridine (m1ψ) and 5-methylcytidine (m5C) modifications in specific regions. We have found that compared with the fully modified mRNAs, the 3′UTR m1ψ modifications alone increased the translation efficiency, and the combined modifications of the m1ψ-3′UTR and m5C-5′UTR/CDS exhibited higher translation efficiency and lower immunogenicity in general. Our study presents a broadly applicable strategy for producing regionally modified mRNAs, advancing the potential of mRNA therapeutics.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"24 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142939656","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}
Gunnar Boysen, Ludmil B Alexandrov, Raheleh Rahbari, Intawat Nookaew, Dave Ussery, Mu-Rong Chao, Chiung-Wen Hu, Marcus S Cooke
Most of the risk factors associated with chronic and complex diseases, such as cancer, stem from exogenous and endogenous exposures experienced throughout an individual’s life, collectively known as the exposome. These exposures can modify DNA, which can subsequently lead to the somatic mutations found in all normal and tumor tissues. Understanding the precise origins of specific somatic mutations has been challenging due to multitude of DNA adducts (i.e. the DNA adductome) and their diverse positions within the genome. Thus far, this limitation has prevented researchers from precisely linking exposures to DNA adducts and DNA adducts to subsequent mutational outcomes. Indeed, many common mutations observed in human cancers appear to originate from error-prone endogenous processes. Consequently, it remains unclear whether these mutations result from exposure-induced DNA adducts, or arise indirectly from endogenous processes or are a combination of both. In this review, we summarize approaches that aim to bridge our understanding of the mechanism by which exposure leads to DNA damage and then to mutation and highlight some of the remaining challenges and shortcomings to fully supporting this paradigm. We emphasize the need to integrate cellular DNA adductomics, long read-based mapping, single-molecule duplex sequencing of native DNA molecules and advanced computational analysis. This proposed holistic approach aims to unveil the causal connections between key DNA modifications and the mutational landscape, whether they originate from external exposures, internal processes or a combination of both, thereby addressing key questions in cancer biology.
{"title":"Investigating the origins of the mutational signatures in cancer","authors":"Gunnar Boysen, Ludmil B Alexandrov, Raheleh Rahbari, Intawat Nookaew, Dave Ussery, Mu-Rong Chao, Chiung-Wen Hu, Marcus S Cooke","doi":"10.1093/nar/gkae1303","DOIUrl":"https://doi.org/10.1093/nar/gkae1303","url":null,"abstract":"Most of the risk factors associated with chronic and complex diseases, such as cancer, stem from exogenous and endogenous exposures experienced throughout an individual’s life, collectively known as the exposome. These exposures can modify DNA, which can subsequently lead to the somatic mutations found in all normal and tumor tissues. Understanding the precise origins of specific somatic mutations has been challenging due to multitude of DNA adducts (i.e. the DNA adductome) and their diverse positions within the genome. Thus far, this limitation has prevented researchers from precisely linking exposures to DNA adducts and DNA adducts to subsequent mutational outcomes. Indeed, many common mutations observed in human cancers appear to originate from error-prone endogenous processes. Consequently, it remains unclear whether these mutations result from exposure-induced DNA adducts, or arise indirectly from endogenous processes or are a combination of both. In this review, we summarize approaches that aim to bridge our understanding of the mechanism by which exposure leads to DNA damage and then to mutation and highlight some of the remaining challenges and shortcomings to fully supporting this paradigm. We emphasize the need to integrate cellular DNA adductomics, long read-based mapping, single-molecule duplex sequencing of native DNA molecules and advanced computational analysis. This proposed holistic approach aims to unveil the causal connections between key DNA modifications and the mutational landscape, whether they originate from external exposures, internal processes or a combination of both, thereby addressing key questions in cancer biology.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"13 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142936963","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}
Huawen Zhong, Wenkai Han, David Gomez-Cabrero, Jesper Tegner, Xin Gao, Guoxin Cui, Manuel Aranda
Cross-species single-cell RNA-seq data hold immense potential for unraveling cell type evolution and transferring knowledge between well-explored and less-studied species. However, challenges arise from interspecific genetic variation, batch effects stemming from experimental discrepancies and inherent individual biological differences. Here, we benchmarked nine data-integration methods across 20 species, encompassing 4.7 million cells, spanning eight phyla and the entire animal taxonomic hierarchy. Our evaluation reveals notable differences between the methods in removing batch effects and preserving biological variance across taxonomic distances. Methods that effectively leverage gene sequence information capture underlying biological variances, while generative model-based approaches excel in batch effect removal. SATURN demonstrates robust performance across diverse taxonomic levels, from cross-genus to cross-phylum, emphasizing its versatility. SAMap excels in integrating species beyond the cross-family level, especially for atlas-level cross-species integration, while scGen shines within or below the cross-class hierarchy. As a result, our analysis offers recommendations and guidelines for selecting suitable integration methods, enhancing cross-species single-cell RNA-seq analyses and advancing algorithm development.
{"title":"Benchmarking cross-species single-cell RNA-seq data integration methods: towards a cell type tree of life","authors":"Huawen Zhong, Wenkai Han, David Gomez-Cabrero, Jesper Tegner, Xin Gao, Guoxin Cui, Manuel Aranda","doi":"10.1093/nar/gkae1316","DOIUrl":"https://doi.org/10.1093/nar/gkae1316","url":null,"abstract":"Cross-species single-cell RNA-seq data hold immense potential for unraveling cell type evolution and transferring knowledge between well-explored and less-studied species. However, challenges arise from interspecific genetic variation, batch effects stemming from experimental discrepancies and inherent individual biological differences. Here, we benchmarked nine data-integration methods across 20 species, encompassing 4.7 million cells, spanning eight phyla and the entire animal taxonomic hierarchy. Our evaluation reveals notable differences between the methods in removing batch effects and preserving biological variance across taxonomic distances. Methods that effectively leverage gene sequence information capture underlying biological variances, while generative model-based approaches excel in batch effect removal. SATURN demonstrates robust performance across diverse taxonomic levels, from cross-genus to cross-phylum, emphasizing its versatility. SAMap excels in integrating species beyond the cross-family level, especially for atlas-level cross-species integration, while scGen shines within or below the cross-class hierarchy. As a result, our analysis offers recommendations and guidelines for selecting suitable integration methods, enhancing cross-species single-cell RNA-seq analyses and advancing algorithm development.","PeriodicalId":19471,"journal":{"name":"Nucleic Acids Research","volume":"204 1","pages":""},"PeriodicalIF":14.9,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142936973","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}
Marina Jordano-Raya, Carol E Schrader, Rafael R Ariza, Teresa Roldán-Arjona, Dolores Córdoba-Cañero
Apurinic/apyrimidinic (AP) endonucleases are key enzymes responsible for the repair of base-less nucleotides generated by spontaneous hydrolysis or as DNA repair intermediates. APE1, the major human AP endonuclease, is a druggable target in cancer and its biological function has been extensively studied. However, the molecular features responsible for its substrate specificity are poorly understood. We show here that, in contrast to APE1, its Arabidopsis ortholog ARP (apurinic endonuclease-redox protein) exhibits orphan base-dependent activity on double-stranded DNA and very poor AP cleavage capacity on single-stranded DNA (ssDNA). We found that these differences are largely a consequence of the variation at two DNA intercalating amino acids that have undergone divergent changes in the metazoan and plant lineages. Swapping the identity of the residue invading the minor groove is sufficient to switch the orphan base specificities of APE1 and ARP. The affinity for ssDNA is largely determined by the major groove invading residue, and swapping its identity switches the ability of APE1 and ARP to cleave AP sites in ssDNA. Importantly, we show that the critical residue for ssDNA cleavage is crucial for mammalian APE1 function in antibody class switch recombination, suggesting an evolutionary advantage for ssDNA activity. These findings provide new molecular insights into the evolution of AP endonucleases.
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