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DNA Origami as Single-Molecule Biosensors
Published in Shuo Huang, Single-Molecule Tools for Bioanalysis, 2022
Travis A. Meyer, Qinyi Lu, Kristin Weiss, Yonggang Ke
In 2014, the Sugiyama group used the DNA origami frame to study site-specific recombination at the single-molecule level, specifically how the orientation of loxP substrates effects the activity of Cre recombinase [55]. In order to study site-specific recombination in real time, a slight change was made in the DNA origami frame design; the anchoring points for loading substrates were positioned such that two perpendicular pairs extended into the center of the frame. Two loxP DNA sequences could be loaded into the frame, such that the ends of the dsDNA were linked to adjacent sides to form loops and duplexes did not overlap with each other. This design allowed the authors to control the orientation of one of the dsDNA segments, in order to compare the differences between loxP arranged in parallel versus antiparallel orientations. AFM imaging of DNA frames following addition of Cre recombinase was used to verify the formation of the synaptic complex (an X-shaped duplex architecture with a bright spot in the center) as well as successful recombination (switch between neighboring anchoring points connected by loops). HS-AFM imaging of recombination events in real time showed that Cre could bind to both antiparallel and parallel loxP orientations but recombination could only occur in the antiparallel conformation. Further changes to substrate orientations enabled studies to investigate preferential cleavage at A-T versus G-C sites as well as effects of preformed intermediate state topology on final resolution.
Structure of revolving biomotors
Published in Peixuan Guo, Zhengyi Zhao, Biomotors: Linear, Rotation, and Revolution Motion Mechanisms, 2017
The FtsK motor contains three functional components: one for DNA translocation, one for orientation control, and one for anchoring itself to the bacterial membrane (Figure 2.2) (Demarre et al., 2013). The N-terminal domain of FtsK consists of four transmembrane helices and interacts with FtsZ, thus leading to its localization and to the forming septum at mid-cell just prior to division (Yu et al., 1998a; Dubarry et al., 2010). FtsK also interacts with a number of downstream cell division proteins to localize them to the divisome and allow cell division to progress (Dubarry et al., 2010). The N and C domains are connected by a linker domain rich in proline and glutamine, which also aids in interactions to localize the cell division apparatus. The C-terminus consists of the DNA translocation motor ATPase, which drives DNA away from the forming septum and also participates in XerCD site-specific recombination at dif sites to resolve chromosome dimers.
Atomic Force Microscope for Topographic Studies
Published in Yuri L. Lyubchenko, An Introduction to Single Molecule Biophysics, 2017
In 1963 Dulbecco and Vogt and Weil and Vinograd made a fundamental discovery. They found that polyoma virus DNA is not a linear but a covalently closed circular molecule. Numerous further studies have led to the conclusion that circular topology rather than linear topology is the predominant DNA form in vivo. There are a number of properties of circular DNA topology, and the sensitivity of the global DNA conformation to the local structural changes is one of them. Local unwinding of the DNA duplex is accompanied by overwinding of the entire circle into а more compact shape termed supercoiled DNA. If the DNA duplex overwinds by n number of turns, this duplex rotation is compensated by interwinding of circular DNA in the opposite direction by the formation of superhelical turns. Importantly, circular plasmid DNA in cells is negatively supercoiled, and this DNA state is tightly linked with various DNA functions. We mention just one; many genetic events require communication between proteins bound to distant sites on DNA including DNA replication, control of gene expression through loop formation, site-specific recombination, and transposition. A fundamental feature of supercoiled DNA geometry is that distantly separated DNA regions can be brought into close proximity (Shlyakhtenko et al. 2000a; Vologodskii 2015). Obviously, direct visualization of such global DNA conformations is important for understanding the biological function of DNA supercoiling. Initially, EM was the only visualization method capable of visualizing supercoiled DNA, but inflexibility of the sample preparation method was a limiting factor in studies focused on labile features such as DNA supercoiling, because the shape of the molecule was very sensitive to such factors as ionic strength and pH. These are not issues for AFM; therefore, this technique was successfully applied to the imaging of supercoiled DNA (Lyubchenko and Shlyakhtenko 1997; Shlyakhtenko et al. 2000a, 2003b; Potaman et al. 2002; Lyubchenko 2004, 2011; Mikheikin et al. 2006; Lyubchenko and Shlyakhtenko 2009).
Predicting algorithm of attC site based on combination optimization strategy
Published in Connection Science, 2022
Zhendong Liu, Xi Chen, Dongyan Li, Xinrong Lv, Mengying Qin, Ke Bai, Zhiqiang He, Yurong Yang, Xiaofeng Li, Qionghai Dai
Gene recombination is a way that organisms use recombinase to recombine different genes to produce new genotype individuals. It is widely present in prokaryotes and has important meanings such as maintaining biological genetic diversity and promoting biological evolution (Epum & Haber, 2022). Common recombination includes: homologous recombination, translocation recombination and site-specific recombination. Currently, with the development of site-specific recombination systems, site-specific recombination technology has been extensively used in various biological genetic engineering operations, especially in higher eukaryotes (Bessen et al., 2019; Häcker et al., 2017). Site-specific recombination refers to the integration, excision and transformation of DNA fragments between specific sites, which is catalysed by integron integrase. This type of recombination is associated with specific DNA sequences in bacteriophages and bacteria, and the reaction always involves two DNA-specific sites. However, these two specific sites usually have very similar or even exactly the same DNA sequences. Such sequence-level constraints restrict efficient recombination between the two sites (Tian & Zhou, 2021). Therefore, in order to solve the problem of sequence constraints, it is necessary to study the structure of specific recombination sites in the recombination system.