DNA Pub Date : 2022-12-01 DOI: 10.3390/dna2040019

Wenyan Xu, Linlin Zhao

{"title":"An Enzyme-Linked Immunosorbent Assay for the Detection of Mitochondrial DNA-Protein Cross-Links from Mammalian Cells.","authors":"Wenyan Xu, Linlin Zhao","doi":"10.3390/dna2040019","DOIUrl":"https://doi.org/10.3390/dna2040019","url":null,"abstract":"DNA-Protein cross-links (DPCs) are cytotoxic DNA lesions with a protein covalently bound to the DNA. Although much has been learned about the formation, repair, and biological consequences of DPCs in the nucleus, little is known regarding mitochondrial DPCs. This is due in part to the lack of robust and specific methods to measure mitochondrial DPCs. Herein, we reported an enzyme-linked immunosorbent assay (ELISA)-based method for detecting mitochondrial DPCs formed between DNA and mitochondrial transcription factor A (TFAM) in cultured human cells. To optimize the purification and detection workflow, we prepared model TFAM-DPCs via Schiff base chemistry using recombinant human TFAM and a DNA substrate containing an abasic (AP) lesion. We optimized the isolation of TFAM-DPCs using commercial silica gel-based columns to achieve a high recovery yield for DPCs. We evaluated the microplate, DNA-coating solution, and HRP substrate for specific and sensitive detection of TFAM-DPCs. Additionally, we optimized the mtDNA isolation procedure to eliminate almost all nuclear DNA contaminants. For proof of concept, we detected the different levels of TFAM-DPCs in mtDNA from HEK293 cells under different biological conditions. The method is based on commercially available materials and can be amended to detect other types of DPCs in mitochondria.","PeriodicalId":72835,"journal":{"name":"DNA","volume":"2 4","pages":"264-278"},"PeriodicalIF":0.0,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10438828/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10047158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 4

Size- and Stereochemistry-Dependent Transcriptional Bypass of DNA Alkyl Phosphotriester Adducts in Mammalian Cells. 哺乳动物细胞中DNA烷基磷酸三酯加合物的大小和立体化学依赖性转录旁路。

DNA Pub Date : 2022-12-01 DOI: 10.3390/dna2040016

Ying Tan, Jiabin Wu, Garrit Clabaugh, Lin Li, Hua Du, Yinsheng Wang

{"title":"Size- and Stereochemistry-Dependent Transcriptional Bypass of DNA Alkyl Phosphotriester Adducts in Mammalian Cells.","authors":"Ying Tan, Jiabin Wu, Garrit Clabaugh, Lin Li, Hua Du, Yinsheng Wang","doi":"10.3390/dna2040016","DOIUrl":"https://doi.org/10.3390/dna2040016","url":null,"abstract":"Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.","PeriodicalId":72835,"journal":{"name":"DNA","volume":"2 4","pages":"221-230"},"PeriodicalIF":0.0,"publicationDate":"2022-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9997456/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9465262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

Mitochondrial DNA: Consensuses and Controversies. 线粒体DNA:共识和争议。

DNA Pub Date : 2022-06-01 Epub Date: 2022-06-10 DOI: 10.3390/dna2020010

Inna Shokolenko, Mikhail Alexeyev

引用次数: 8

Nucleases and Co-Factors in DNA Replication Stress Responses. DNA复制应激反应中的核酸酶和共因子。

DNA Pub Date : 2022-03-01 DOI: 10.3390/dna2010006

Jac A Nickoloff, Neelam Sharma, Lynn Taylor, Sage J Allen, Robert Hromas

{"title":"Nucleases and Co-Factors in DNA Replication Stress Responses.","authors":"Jac A Nickoloff, Neelam Sharma, Lynn Taylor, Sage J Allen, Robert Hromas","doi":"10.3390/dna2010006","DOIUrl":"10.3390/dna2010006","url":null,"abstract":"DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics.","PeriodicalId":72835,"journal":{"name":"DNA","volume":"2 1","pages":"68-85"},"PeriodicalIF":0.0,"publicationDate":"2022-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9534323/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33492845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Small RNAs Worm Up Transgenerational Epigenetics Research. 小核糖核酸催生跨代表观遗传学研究

DNA Pub Date : 2021-12-01 Epub Date: 2021-09-29 DOI: 10.3390/dna1020005

Alla Grishok

引用次数: 0

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