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1. Modeling RNA:DNA Hybrids with Formal Grammars

   https://doi.org/10.1007/978-3-030-57129-0_3  

   Jonoska, N. ; Obatake, N. ; Poznanović, S. ; Price, C. ; Riehl, M. ; Vazquez, M. ( January 2020 , Using Mathematics to Understand Biological Complexity)

   Segal, R. ; Shtylla, B. ; Sindi, S (Ed.)

   R-loops are nucleic acid structures consisting of a DNA:RNA hybrid and a DNA single strand. They form naturally during transcription when the nascent RNA hybridizes to the template DNA, forcing the coding DNA strand to wrap around the RNA:DNA duplex. Although formation of R-loops can have deleterious effects on genome integrity, there is evidence of their role as potential regulators of gene expression and DNA repair. Here we initiate an abstract model based on formal grammars to describe RNA:DNA interactions and the formation of R-loops. Separately we use a sliding window approach that accounts for properties of the DNA nucleotide sequence, such as C-richness and CG-skew, to identify segments favoring R-loops. We evaluate these properties on two DNA plasmids that are known to form R-loops and compare results with a recent energetics model from the Chédin Lab. Our abstract approach for R-loops is an initial step toward a more sophisticated framework which can take into account the effect of DNA topology on R-loop formation. 

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2. Interplay between DNA sequence and negative superhelicity drives R-loop structures

   https://doi.org/10.1073/pnas.1819476116  

   Stolz, Robert ; Sulthana, Shaheen ; Hartono, Stella R. ; Malig, Maika ; Benham, Craig J. ; Chedin, Frederic ( March 2019 , Proceedings of the National Academy of Sciences)

   R-loops are abundant three-stranded nucleic-acid structures that formin cisduring transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms—junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

    

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3. R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline

   https://doi.org/10.1093/nar/gkac621  

   Hicks, Tara ; Koury, Emily ; McCabe, Caleb ; Williams, Cameron ; Crahan, Caroline ; Smolikove, Sarit ( July 2022 , Nucleic Acids Research)

   Accumulation of DNA–RNA hybrids in the form of R-loops can result in replication–transcription conflict that leads to the formation of DNA double strand breaks (DSBs). Using null mutants for the two Caenorhabditis elegans genes encoding for RNaseH1 and RNaseH2, we identify novel effects of R-loop accumulation in the germline. R-loop accumulation leads, as expected, to replication stress, followed by the formation of DSBs. A subset of these DSBs are irreparable. However, unlike irreparable DSBs generated in other systems, which trigger permanent cell cycle arrest, germline irreparable DSBs are propagated to oocytes. Despite DNA damage checkpoint activation in the stem cell niche, the signaling cannot be sustained and nuclei with irreparable DNA damage progress into meiosis. Moreover, unlike other forms of DNA damage that increase germline apoptosis, R-loop-generated DSBs remain undetected by the apoptotic checkpoint. This coincides with attenuation of ATM/ATR signaling in mid-to-late meiotic prophase I. These data altogether indicate that in the germline, DSBs that are generated by R-loops can lead to irreparable DSBs that evade cellular machineries designed for damage recognition. These studies implicate germline R-loops as an especially dangerous driver of germline mutagenesis.

    

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4. Crystal structure of an RNA/DNA strand exchange junction

   https://doi.org/10.1371/journal.pone.0263547  

   Cofsky, Joshua C. ; Knott, Gavin J. ; Gee, Christine L. ; Doudna, Jennifer A. ( April 2022 , PLOS ONE)

   Kursula, Petri (Ed.)

   Short segments of RNA displace one strand of a DNA duplex during diverse processes including transcription and CRISPR-mediated immunity and genome editing. These strand exchange events involve the intersection of two geometrically distinct helix types—an RNA:DNA hybrid (A-form) and a DNA:DNA homoduplex (B-form). Although previous evidence suggests that these two helices can stack on each other, it is unknown what local geometric adjustments could enable A-on-B stacking. Here we report the X-ray crystal structure of an RNA-5′/DNA-3′ strand exchange junction at an anisotropic resolution of 1.6 to 2.2 Å. The structure reveals that the A-to-B helical transition involves a combination of helical axis misalignment, helical axis tilting and compression of the DNA strand within the RNA:DNA helix, where nucleotides exhibit a mixture of A- and B-form geometry. These structural principles explain previous observations of conformational stability in RNA/DNA exchange junctions, enabling a nucleic acid architecture that is repeatedly populated during biological strand exchange events. 

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5. CRISPR–Cas molecular beacons as tool for studies of assembly of CRISPR–Cas effector complexes and their interactions with DNA

   Mekler, Vladamir ; Kuznedelov, Konstantin ; Minakhin, Leonid ; Murugan, Karthik ; Sashital, Dipali G. ; Severinov, Konstantin ( January 2019 , Methods in enzymology)

   CRISPR–Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR–Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR–Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. The DNA interrogation and R-loop formation involve several distinct steps the molecular details of which are not fully understood. This chapter describes a recently developed fluorometric Cas beacon assay that may be used for measuring of specific affinity of various CRISPR–Cas complexes for unlabeled target DNA and model DNA probes. The Cas beacon approach also can provide a sensitive method for monitoring the kinetics of assembly of CRISPR–Cas complexes. 

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