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1. Deciphering the protein‐DNA code of bacterial winged helix‐turn‐helix transcription factors

   https://doi.org/10.1007/s40484-018-0130-0  

   Joyce, Adam P. ; Havranek, James J. ( March 2018 , Quantitative Biology)

   Sequence‐specific binding by transcription factors (TFs) plays a significant role in the selection and regulation of target genes. At the protein:DNA interface, amino acid side‐chains construct a diverse physicochemical network of specific and non‐specific interactions, and seemingly subtle changes in amino acid identity at certain positions may dramatically impact TF:DNA binding. Variation of these specificity‐determining residues (SDRs) is a major mechanism of functional divergence between TFs with strong structural or sequence homology.

   In this study, we employed a combination of high‐throughput specificity profiling by SELEX and Spec‐seq, structural modeling, and evolutionary analysis to probe the binding preferences of winged helix‐turn‐helix TFs belonging to the OmpR sub‐family inEscherichia coli.

   We found thatE. coliOmpR paralogs recognize tandem, variably spaced repeats composed of “GT‐A” or “GCT”‐containing half‐sites. Some divergent sequence preferences observed within the “GT‐A” mode correlate with amino acid similarity; conversely, “GCT”‐based motifs were observed for a subset of paralogs with low sequence homology. Direct specificity profiling of a subset of OmpR homologues (CpxR, RstA, and OmpR) as well as predicted “SDR‐swap” variants revealed that individual SDRs may impact sequence preferences locally through direct contact with DNA bases or distally via the DNA backbone.

   Overall, our work provides evidence for a common structural “code” for sequence‐specific wHTH‐DNA interactions, and demonstrates that surprisingly modest residue changes can enable recognition of highly divergent sequence motifs. Further examination of SDR predictions will likely reveal additional mechanisms controlling the evolutionary divergence of this important class of transcriptional regulators.

    

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2. The bridge helix of Cas12a imparts selectivity in cis ‐DNA cleavage and regulates trans ‐DNA cleavage

   https://doi.org/10.1002/1873-3468.14051  

   Parameshwaran, Hari Priya ; Babu, Kesavan ; Tran, Christine ; Guan, Kevin ; Allen, Aleique ; Kathiresan, Venkatesan ; Qin, Peter Z. ; Rajan, Rakhi ( February 2021 , FEBS Letters)

   Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity inStreptococcus pyogenes(Spy) Cas9. Here, we examined a BH variant of Cas12a fromFrancisella novicida(FnoCas12a^KD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12a^KD2Paccumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12a^KD2Palso possess reducedtrans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in bothcis‐andtrans‐cleavages and show its conserved role in target discrimination among Cas nucleases.

    

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3. Bridge Helix of Cas9 Modulates Target DNA Cleavage and Mismatch Tolerance

   https://doi.org/10.1021/acs.biochem.8b01241  

   Babu, Kesavan ; Amrani, Nadia ; Jiang, Wei ; Yogesha, S.D. ; Nguyen, Richard ; Qin, Peter Z. ; Rajan, Rakhi ( March 2019 , Biochemistry)
4. Chiral Sum Frequency Generation Spectroscopy Detects Double-Helix DNA at Interfaces

   https://doi.org/10.1021/acs.langmuir.2c00365  

   Perets, Ethan A. ; Olesen, Kristian B. ; Yan, Elsa C. ( April 2022 , Langmuir)
5. DNA double helix, a tiny electromotor

   https://doi.org/10.1038/s41565-022-01285-z  

   Maffeo, Christopher ; Quednau, Lauren ; Wilson, James ; Aksimentiev, Aleksei ( December 2022 , Nature Nanotechnology)

   Flowing fluid past chiral objects has been used for centuries to power rotary motion in man-made machines. By contrast, rotary motion in nanoscale biological or chemical systems is produced by biasing Brownian motion through cyclic chemical reactions. Here we show that a chiral biological molecule, a DNA or RNA duplex rotates unidirectionally at billions of revolutions per minute when an electric field is applied along the duplex, with the rotation direction being determined by the chirality of the duplex. The rotation is found to be powered by the drag force of the electro-osmotic flow, realizing the operating principle of a macroscopic turbine at the nanoscale. The resulting torques are sufficient to power rotation of nanoscale beads and rods, offering an engineering principle for constructing nanoscale systems powered by electric field. 

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