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1. Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

   https://doi.org/10.1093/nar/gkaa1267  

   Naufer, M Nabuan ; Morse, Michael ; Möller, Guðfríður Björg ; McIsaac, James ; Rouzina, Ioulia ; Beuning, Penny J ; Williams, Mark C ( January 2021 , Nucleic Acids Research)

   null (Ed.)

   Abstract Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations. 

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2. Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2′‐deoxy‐ATP

   https://doi.org/10.1113/JP280402  

   Ma, Weikang ; Childers, Matthew ; Murray, Jason ; Moussavi‐Harami, Farid ; Gong, Henry ; Weiss, Robert ; Daggett, Valerie ; Irving, Thomas ; Regnier, Michael ( September 2020 , The Journal of Physiology)

   Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.

   The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.

   X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.

   Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.

   This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases.

   Here we used time‐resolved small‐angle X‐ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first‐order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (S_M3and S_M6) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2‐deoxy‐ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X‐ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin‐binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

    

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3. HRB2 and BBX21 interaction modulates Arabidopsis ABI5 locus and stomatal aperture

   https://doi.org/10.1111/pce.13336  

   Kang, Xiaojun ; Xu, Gang ; Lee, Byungha ; Chen, Chen ; Zhang, Huanan ; Kuang, Rui ; Ni, Min ( May 2018 , Plant, Cell & Environment)

   Blue light triggers the opening of stomata in the morning to allow CO₂uptake and water loss through transpiration. During the day, plants may experience periodic drought and accumulate abscisic acid (ABA). ABA antagonizes blue light signalling through phosphatidic acid and reduces stomatal aperture. This study reveals a molecular mechanism by which two light signalling proteins interact to repress ABA signalling in the control of stomatal aperture. Ahypersensitive to red and blue 2(hrb2) mutant has a defective ATP‐dependent chromatin‐remodelling factor, PKL, in the chromodomain/helicase/DNA binding family. HRB2 enhances the light‐induced expression of a B‐box transcription factor gene,BBX21. BBX21 binds a T/G box in theABI5promoter and recruits HRB2 to modulate the chromatin structure at theABI5locus. Mutation in eitherHRB2orBBX21led to reduced water loss and ABA hypersensitivity. This hypersensitivity to ABA was well explained by the enhanced expression of the ABA signalling geneABI5in both mutants. Indeed, stomatal aperture was significantly reduced byABI5overexpression in the absence or presence of ABA under monochromatic light conditions. Overall, we present a regulatory loop in which two light signalling proteins repress ABA signalling to sustain gas exchange when plants experience periodic drought.

    

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4. Interaction with single‐stranded DNA‐binding protein localizes ribonuclease HI to DNA replication forks and facilitates R‐loop removal

   https://doi.org/10.1111/mmi.14529  

   Wolak, Christine ; Ma, Hui Jun ; Soubry, Nicolas ; Sandler, Steven J. ; Reyes‐Lamothe, Rodrigo ; Keck, James L. ( June 2020 , Molecular Microbiology)

   DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R‐loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R‐loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R‐loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction betweenEscherichia coliRNase HI and the single‐stranded DNA‐binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation ofrnhAto encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media‐dependent slow‐growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R‐loop accumulation enhance or suppress, respectively, the growth phenotype ofrnhAK60E rep::kanstrains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R‐loops that block DNA replication.

    

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5. A single nucleobase tunes nonradiative decay in a DNA-bound silver cluster

   https://doi.org/10.1063/5.0056836  

   Zhang, Yuyuan ; He, Chen ; de La Harpe, Kimberly ; Goodwin, Peter M. ; Petty, Jeffrey T. ; Kohler, Bern ( September 2021 , The Journal of Chemical Physics)

   DNA strands are polymeric ligands that both protect and tune molecular-sized silver cluster chromophores. We studied single-stranded DNA C4AC4TC3XT4 with X = guanosine and inosine that form a green fluorescent Ag106+ cluster, but these two hosts are distinguished by their binding sites and the brightness of their Ag106+ adducts. The nucleobase subunits in these oligomers collectively coordinate this cluster, and fs time-resolved infrared spectra previously identified one point of contact between the C2–NH2 of the X = guanosine, an interaction that is precluded for inosine. Furthermore, this single nucleobase controls the cluster fluorescence as the X = guanosine complex is ∼2.5× dimmer. We discuss the electronic relaxation in these two complexes using transient absorption spectroscopy in the time window 200 fs–400 µs. Three prominent features emerged: a ground state bleach, an excited state absorption, and a stimulated emission. Stimulated emission at the earliest delay time (200 fs) suggests that the emissive state is populated promptly following photoexcitation. Concurrently, the excited state decays and the ground state recovers, and these changes are ∼2× faster for the X = guanosine compared to the X = inosine cluster, paralleling their brightness difference. In contrast to similar radiative decay rates, the nonradiative decay rate is 7× higher with the X = guanosine vs inosine strand. A minor decay channel via a dark state is discussed. The possible correlation between the nonradiative decay and selective coordination with the X = guanosine/inosine suggests that specific nucleobase subunits within a DNA strand can modulate cluster–ligand interactions and, in turn, cluster brightness. 

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