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1. Agrobacterium T‐DNA integration in somatic cells does not require the activity of DNA polymerase θ

   https://doi.org/10.1111/nph.17032  

   Nishizawa‐Yokoi, Ayako; Saika, Hiroaki; Hara, Naho; Lee, Lan‐Ying; Toki, Seiichi; Gelvin, Stanton B. (December 2020, New Phytologist)

   Summary Integration ofAgrobacterium tumefacienstransferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type andpolQmutantArabidopsisand rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway. 

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2. Rationally Programming Nanomaterials with DNA for Biomedical Applications

   https://doi.org/10.1002/advs.202003775  

   He, Liangcan; Mu, Jing; Gang, Oleg; Chen, Xiaoyuan (February 2021, Advanced Science)

   Abstract DNA is not only a carrier of genetic information, but also a versatile structural tool for the engineering and self‐assembling of nanostructures. In this regard, the DNA template has dramatically enhanced the scalability, programmability, and functionality of the self‐assembled DNA nanostructures. These capabilities provide opportunities for a wide range of biomedical applications in biosensing, bioimaging, drug delivery, and disease therapy. In this review, the importance and advantages of DNA for programming and fabricating of DNA nanostructures are first highlighted. The recent progress in design and construction of DNA nanostructures are then summarized, including DNA conjugated nanoparticle systems, DNA‐based clusters and extended organizations, and DNA origami‐templated assemblies. An overview on biomedical applications of the self‐assembled DNA nanostructures is provided. Finally, the conclusion and perspectives on the self‐assembled DNA nanostructures are presented. 

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3. The Escherichia coli clamp loader rapidly remodels SSB on DNA to load clamps

   https://doi.org/10.1093/nar/gkac1169  

   Newcomb, Elijah S. P.; Douma, Lauren G.; Morris, Leslie A.; Bloom, Linda B. (December 2022, Nucleic Acids Research)

   Abstract Single-stranded DNA binding proteins (SSBs) avidly bind ssDNA and yet enzymes that need to act during DNA replication and repair are not generally impeded by SSB, and are often stimulated by SSB. Here, the effects of Escherichia coli SSB on the activities of the DNA polymerase processivity clamp loader were investigated. SSB enhances binding of the clamp loader to DNA by increasing the lifetime on DNA. Clamp loading was measured on DNA substrates that differed in length of ssDNA overhangs to permit SSB binding in different binding modes. Even though SSB binds DNA adjacent to single-stranded/double-stranded DNA junctions where clamps are loaded, the rate of clamp loading on DNA was not affected by SSB on any of the DNA substrates. Direct measurements of the relative timing of DNA-SSB remodeling and enzyme–DNA binding showed that the clamp loader rapidly remodels SSB on DNA such that SSB has little effect on DNA binding rates. However, when SSB was mutated to reduce protein–protein interactions with the clamp loader, clamp loading was inhibited by impeding binding of the clamp loader to DNA. Thus, protein–protein interactions between the clamp loader and SSB facilitate rapid DNA-SSB remodeling to allow rapid clamp loader-DNA binding and clamp loading. 

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4. Ethanol preservation and pretreatments facilitate quality DNA extractions in recalcitrant plant species

   https://doi.org/10.1002/aps3.11519  

   Johnson, Gabriel; Canty, Steven W.; Lichter‐Marck, Isaac H.; Wagner, Warren; Wen, Jun (May 2023, Applications in Plant Sciences)

   Abstract PremiseThe preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high‐quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples. MethodsDNA was isolated from leaves preserved with 96% ethanol or from silica‐desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method. ResultsDNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol‐pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples. DiscussionThis study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies. 

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5. Bridging DNA contacts allow Dps from E. coli to condense DNA

   https://doi.org/10.1093/nar/gkae223  

   Shahu, Sneha; Vtyurina, Natalia; Das, Moumita; Meyer, Anne S.; Ganji, Mahipal; Abbondanzieri, Elio A. (April 2024, Nucleic Acids Research)

   Abstract The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps–DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes. 

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