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1. Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness

   https://doi.org/10.1371/journal.pgen.1009886  

   Behrmann, Megan S. ; Perera, Himasha M. ; Hoang, Joy M. ; Venkat, Trisha A. ; Visser, Bryan J. ; Bates, David ; Trakselis, Michael A. ( November 2021 , PLOS Genetics)

   Reyes Lamothe, Rodrigo (Ed.)

   Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro . To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB : mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo , supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork. 

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2. Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

   https://doi.org/10.7554/eLife.34186  

   Ma, Wen ; Whitley, Kevin D ; Chemla, Yann R ; Luthey-Schulten, Zaida ; Schulten, Klaus ( April 2018 , eLife)

   Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines. 

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3. RadD is a RecA-dependent accessory protein that accelerates DNA strand exchange

   https://doi.org/10.1093/nar/gkac041  

   Bonde, Nina J. ; Romero, Zachary J. ; Chitteni-Pattu, Sindhu ; Cox, Michael M. ( February 2022 , Nucleic Acids Research)

   In rapidly growing cells, with recombinational DNA repair required often and a new replication fork passing every 20 min, the pace of RecA-mediated DNA strand exchange is potentially much too slow for bacterial DNA metabolism. The enigmatic RadD protein, a putative SF2 family helicase, exhibits no independent helicase activity on branched DNAs. Instead, RadD greatly accelerates RecA-mediated DNA strand exchange, functioning only when RecA protein is present. The RadD reaction requires the RadD ATPase activity, does not require an interaction with SSB, and may disassemble RecA filaments as it functions. We present RadD as a new class of enzyme, an accessory protein that accelerates DNA strand exchange, possibly with a helicase-like action, in a reaction that is entirely RecA-dependent. RadD is thus a DNA strand exchange (recombination) synergist whose primary function is to coordinate closely with and accelerate the DNA strand exchange reactions promoted by the RecA recombinase. Multiple observations indicate a uniquely close coordination of RadD with RecA function.

    

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4. Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli

   https://doi.org/10.1534/g3.120.401106  

   Klaric, Julie A. ; Perr, Eli L. ; Lovett, Susan T. ( May 2020 , G3: Genes|Genomes|Genetics)

   DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations. 

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5. Wss1 homolog from Candida albicans and its role in DNA–protein crosslink tolerance

   https://doi.org/10.1111/mmi.14518  

   Homchan, Aimorn ; Sukted, Juthamas ; Mongkolsuk, Skorn ; Jeruzalmi, David ; Matangkasombut, Oranart ; Pakotiprapha, Danaya ( May 2020 , Molecular Microbiology)

   Candida albicansis an opportunistic yeast that can cause life‐threatening systemic infection in immunocompromised individuals. During infections,C. albicanshas to cope with genotoxic stresses generated by the host immune system. DNA–protein crosslink (DPC), the covalent linkage of proteins with DNA, is one type of DNA damages that can be caused by the host immune response. DPCs are bulky lesions that interfere with the progression of replication and transcription machineries, and hence threaten genomic integrity. Accordingly, either a DPC tolerance mechanism or a DPC repair pathway is essential forC. albicansto maintain genomic stability and survive in the host. Here, we identified Wss1 (weak suppressor of Smt3) inC. albicans(CaWss1) using bioinformatics, genetic complementation, and biochemical studies. We showed thatCaWss1 promotes cell survival under genotoxic stress conditions that generate DPCs and that the catalytic metalloprotease domain ofCaWss1 is essential for its cellular function. Interactions ofCaWss1 with Cdc48 and small ubiquitin‐like modifier, although not strictly required, contribute to the function ofCaWss1 in the suppression of the growth defects under DPC‐inducing conditions. This report is the first investigation of the role ofCaWss1 in DPC tolerance inC. albicans.

    

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