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1. Mono- and bilayer smectic liquid crystal ordering in dense solutions of “gapped” DNA duplexes

   https://doi.org/10.1073/pnas.2019996118  

   Gyawali, Prabesh; Saha, Rony; Smith, Gregory P.; Salamonczyk, Miroslaw; Kharel, Prakash; Basu, Soumitra; Li, Ruipeng; Fukuto, Masafumi; Gleeson, James T.; Clark, Noel A.; et al (March 2021, Proceedings of the National Academy of Sciences)

   Although its mesomorphic properties have been studied for many years, only recently has the molecule of life begun to reveal the true range of its rich liquid crystalline behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes—driving the self-assembly of aggregates that organize into liquid crystal phases—and the incorporation of flexible single-stranded “gaps” in otherwise fully paired duplexes—producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions—are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic ordering in concentrated solutions of various “gapped” DNA (GDNA) constructs. We examine symmetric GDNA constructs consisting of two 48-base pair duplex segments bridged by a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range $\sim 230\text{to}\sim 280$mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths (“bilayer” structure), and the other has a period similar to a single-duplex length (“monolayer” structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to $>40\hspace{0.17em}°$C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures. 

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2. Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing

   https://doi.org/10.1038/s41467-022-35572-z  

   Caldwell, Brian J.; Norris, Andrew S.; Karbowski, Caroline F.; Wiegand, Alyssa M.; Wysocki, Vicki H.; Bell, Charles E. (December 2022, Nature Communications)

   Abstract Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redβ from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redβ homolog from a prophage ofListeria innocuain complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA. 

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3. Weak tension accelerates hybridization and dehybridization of short oligonucleotides

   https://doi.org/10.1093/nar/gkad118  

   Hart, Derek J; Jeong, Jiyoun; Gumbart, James C; Kim, Harold D (March 2023, Nucleic Acids Research)

   Abstract The hybridization and dehybridization of DNA subject to tension is relevant to fundamental genetic processes and to the design of DNA-based mechanobiology assays. While strong tension accelerates DNA melting and decelerates DNA annealing, the effects of tension weaker than 5 pN are less clear. In this study, we developed a DNA bow assay, which uses the bending rigidity of double-stranded DNA (dsDNA) to exert weak tension on a single-stranded DNA (ssDNA) target in the range of 2–6 pN. Combining this assay with single-molecule FRET, we measured the hybridization and dehybridization kinetics between a 15 nt ssDNA under tension and a 8–9  nt oligonucleotide, and found that both the hybridization and dehybridization rates monotonically increase with tension for various nucleotide sequences tested. These findings suggest that the nucleated duplex in its transition state is more extended than the pure dsDNA or ssDNA counterpart. Based on coarse-grained oxDNA simulations, we propose that this increased extension of the transition state is due to steric repulsion between the unpaired ssDNA segments in close proximity to one another. Using linear force-extension relations verified by simulations of short DNA segments, we derived analytical equations for force-to-rate conversion that are in good agreement with our measurements. 

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4. 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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5. Eukaryotic Circular Rep-Encoding Single-Stranded DNA (CRESS DNA) Viruses: Ubiquitous Viruses With Small Genomes and a Diverse Host Range

   Zhao, L; Rosario, K; Breitbart, M; Duffy, S. (January 2019, Advances in virus research)

   While single-stranded DNA (ssDNA) was once thought to be a relatively rare genomic architecture for viruses, modern metagenomics sequencing has revealed circular ssDNA viruses in most environments and in association with diverse hosts. In particular, circular ssDNA viruses encoding a homologous replication-associated protein (Rep) have been identified in the majority of eukaryotic supergroups, generating interest in the ecological effects and evolutionary history of circular Rep-encoding ssDNA viruses (CRESS DNA) viruses. This review surveys the explosion of sequence diversity and expansion of eukaryotic CRESS DNA taxonomic groups over the last decade, highlights similarities between the well-studied geminiviruses and circoviruses with newly identified groups known only through their genome sequences, discusses the ecology and evolution of eukaryotic CRESS DNA viruses, and speculates on future research horizons. 

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